Dosing regimens for use with pcsk9 inhibitors

ABSTRACT

The present invention provides methods for treating a PCSK9-mediated disease or a PCSK9-mediated condition. Specifically, the invention relates to methods comprising the administration of a proprotein convertase subtilisin/kexin type 9 (PCSK9) antibody or antigen binding protein, in the absence of a statin, to a subject in need thereof.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/415,837, filed May 17, 2019, which is a continuation of U.S. patentapplication Ser. No. 14/539,199, filed Nov. 12, 2014, now U.S. Pat. No.10,428,157, which claims priority to U.S. Provisional Patent ApplicationNo. 61/902,857, filed Nov. 12, 2013, U.S. Provisional Patent ApplicationNo. 61/955,337, filed Mar. 19, 2014, and European Patent Application No.14306222.2, filed Jul. 31, 2014, the contents of each of which areincorporated herein by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in XML format and is hereby incorporated byreference in its entirety. Said XML file, created on Apr. 17, 2023, isnamed 741929_SA9-132CON2_ST26.xml and is 229,244 bytes in size.

FIELD OF THE INVENTION

The present invention relates to the field of therapeutic treatment fora PCSK9-mediated disease or a PCSK9-mediated condition. Specifically,the invention relates to methods comprising the administration of aproprotein convertase subtilisin/kexin type 9 (PCSK9) antagonist, e.g.,an anti-PCSK9 antibody or antigen binding protein, in the absence of astatin to a subject in need thereof. The invention also relates tomethods comprising the administration of a high dose, low frequencydosing regimen of a PCSK9 antibody or antigen binding protein to asubject who is not taking a concomitant statin.

BACKGROUND

Hypercholesterolemia, particularly an increase in low-densitylipoprotein cholesterol (LDL-C) levels, constitutes a major risk for thedevelopment of atherosclerosis and coronary heart disease (CHD), theleading cause of death and disability in the Western world. Numerousstudies have demonstrated that reducing LDL-C levels, mainly with3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG CoA) inhibitors (commonlyreferred to as statins), reduces the risk of CHD, with a strong directrelationship between LDL-C levels and CHD events; for each 1 mmol/L (˜40mg/dL) reduction in LDL-C, cardiovascular disease (CVD) mortality andmorbidity is lowered by 22%. Greater reductions in LDL-C produce greaterreduction in events, and comparative data of intensive versus standardstatin treatment suggest that the lower the LDL-C level, the greater thebenefit in patients at high cardiovascular risk.

The long-term elevations of LDL-C leading to a progressive accumulationof coronary atherosclerosis require a long-term management, whichincludes lifestyle measures as the primary intervention. However, sincelifestyle measures rarely reduce plasma LDL-C by >15%, use ofpharmacologic treatments are needed to adequately treat dyslipidemicpatients. Current LDL-C lowering medications include statins, ezetimibe(EZE), fibrates, niacin, and bile acid sequestrants, of which statinsare the most commonly prescribed, as they have shown a great ability tolower LDL-C and reduce CHD events. Since hypercholesterolemia is largelyasymptomatic, side effects of pharmacologic agents used to manage it canundermine patient compliance. In several cohort studies, the reportedrate of adherence to statin therapy at 1 year ranged from 26% to 85%,with a rapid decline in adherence rates typically observed within thefirst few months.

Despite the widespread availability of lipid-modifying therapies, suchas statins, approximately 30% of all adult patients treated forhypercholesterolemia in the United States between 1999 and 2006 failedto achieve their recommended LDL-C targets. Reasons for this includepoor adherence to therapy, drug-resistance/intolerance, and the positiverelationship between adverse event rates and increasing dosage.Moreover, since the most effective lipid-modifying therapies can onlyreduce LDL-C levels by up to 55%, target attainment rates in patientsthat require substantial reductions in LDL-C, such as those withfamilial hypercholesterolemia, are often significantly lower than mightbe expected. More effective lipid-modifying therapies and treatmentregimens are therefore required to improve target attainment rates inthese patients.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a proproteinconvertase belonging to the proteinase K subfamily of the secretorysubtilase family. Evidence suggests that PCSK9 increases plasma LDLcholesterol by promoting degradation of the LDL receptor, which mediatesLDL endocytosis in the liver, the major route of LDL clearance fromcirculation.

The use of PCSK9 inhibitors (anti-PCSK9 antibodies) to reduce serumtotal cholesterol, LDL cholesterol, and serum triglycerides has beendescribed in U.S. Pat. Nos. 8,062,640 and 8,357,371, and U.S. PatentApplication Publication No. 2013/0064834. However, there remains a needin the art for improved therapeutic methods.

BRIEF SUMMARY OF THE INVENTION

The present invention addresses the need in the art for improvedtherapeutic methods by establishing the beneficial effects ofadministering a PCSK9 antibody or antigen binding protein to a subjectin the absence of a statin at a lower frequency. The present inventionprovides methods for treating hypercholesterolemia and/or reducingLDL-cholesterol by administering a high dose, low frequency dosingregimen of an anti-PCSK9 antibody, in the absence of statin.

One embodiment provides a method for reducing low-density lipoproteincholesterol (LDL-C) in a subject in need thereof by administering to thesubject, who is not taking a concomitant statin, a pharmaceuticalcomposition comprising an anti-proprotein convertase subtilisin/kexintype 9 (anti-PCSK9) antibody or antigen-binding protein at a dose ofabout 150 mg every 4 weeks for at least 3 doses, thereby reducing theLDL-C in the subject.

Another embodiment provides a method for treating hypercholesterolemiain a subject in need thereof by administering to the subject, who is nottaking a concomitant statin, a pharmaceutical composition comprising ananti-proprotein convertase subtilisin/kexin type 9 (anti-PCSK9) antibodyor antigen-binding protein at a dose of about 150 mg every 4 weeks forat least 3 doses, thereby treating the hypercholesterolemia in thesubject.

One embodiment provides a method for maintaining constant low-densitylipoprotein cholesterol (LDL-C) lowering throughout an interdosinginterval in a subject by administering to the subject, who is not takinga concomitant statin, a pharmaceutical composition comprising ananti-proprotein convertase subtilisin/kexin type 9 (anti-PCSK9) antibodyor antigen-binding protein at a dose of about 150 mg every 4 weeks forat least 3 doses. The invention also provides a method for increasingthe duration of action of a proprotein convertase subtilisin/kexin type9 (PCSK9) antagonist in a subject by administering to the subject aPCSK9 antagonist in the absence of a statin.

In some embodiments, the subject has heterozygous FamilialHypercholesterolemia (heFH). In other embodiments, the subject has aform of hypercholesterolemia that is not Familial Hypercholesterolemia(nonFH).

The present invention also includes a method for treating a form ofhypercholesterolemia that is not Familial Hypercholesterolemia in asubject in need thereof by administering to the subject a pharmaceuticalcomposition comprising an anti-proprotein convertase subtilisin/kexintype 9 (anti-PCSK9) antibody or antigen-binding protein at a dose ofabout 150 mg every 4 weeks for at least 3 doses, thereby treating theform of hypercholesterolemia that is not Familial Hypercholesterolemiain the subject.

In some embodiments, subject is on a non-statin lipid-lowering agentbefore and/or during administration of the antibody or antigen-bindingprotein. In some embodiments, the non-statin lipid-lowering agent isselected from the group consisting of: ezetimibe, a fibrate,fenofibrate, niacin, an omega-3 fatty acid, and a bile acid resin. Inspecific embodiments, the non-statin lipid-lowering agent is ezetimibeor fenofibrate. In other embodiments, the subject is not on a non-statinlipid-lowering agent before and/or during administration of the antibodyor antigen-binding protein.

In some embodiments, the PCSK9 antibody or antigen-binding fragmentthereof comprises the heavy and light chain complementarity determiningregions (CDRs) of a heavy chain variable region/light chain variableregion (HCVR/LCVR) amino acid sequence pair selected from the groupconsisting of SEQ ID NOs: 1/6 and 11/15. In some aspects, the antibodyor antigen-binding fragment thereof comprises heavy and light chain CDRamino acid sequences having SEQ ID NOs:12, 13, 14, 16, 17, and 18. Insome aspects, the antibody or antigen-binding fragment thereof comprisesan HCVR having the amino acid sequence of SEQ ID NO:11 and an LCVRhaving the amino acid sequence of SEQ ID NO:15. In some aspects, theantibody or antigen-binding fragment thereof comprises heavy and lightchain CDR amino acid sequences having SEQ ID NOs:2, 3, 4, 7, 8, and 10.In some aspects, the antibody or antigen-binding fragment thereofcomprises an HCVR having the amino acid sequence of SEQ ID NO:1 and anLCVR having the amino acid sequence of SEQ ID NO:6.

In certain aspects of the invention, the antibody or antigen-bindingfragment thereof binds to the same epitope on PCSK9 as an antibodycomprising heavy and light chain CDR amino acid sequences having SEQ IDNOs:12, 13, 14, 16, 17, and 18; or SEQ ID NOs: 2, 3, 4, 7, 8, and 10.

In certain aspects of the invention, the antibody or antigen-bindingfragment thereof competes for binding to PCSK9 with an antibodycomprising heavy and light chain CDR amino acid sequences having SEQ IDNOs:12, 13, 14, 16, 17, and 18; or SEQ ID NOs:2, 3, 4, 7, 8, and 10.

The methods of the invention include administering an anti-PCSK9antibody or antigen-binding protein to a subject at a dose of about 150mg every 4 weeks for at least three doses. In certain embodiments, theantibody is administered at a dose of about 150 mg every four weeks forthree doses, and the dose remains at about 150 mg every four weeks ifthe subject's LDL-C value is less than a target LDL-C level. In someembodiments, the LDL-C of the subject is measured twelve weeks after thesubject received the first dose of the antibody or antigen-bindingprotein. In some embodiments, the target LDL-C level is less than 70milligrams per deciliter (mg/dL). In alternative embodiments, the targetLDL-C level is less than 70 milligrams per deciliter (mg/dL) and a 30%reduction of LDL-C. In other embodiments, the target LDL-C level is lessthan 100 milligrams per deciliter (mg/dL) In alternative embodiments,the target LDL-C level is less than 100 milligrams per deciliter (mg/dL)and a 30% reduction of LDL-C.

The methods of the invention include administering an anti-PCSK9antibody or antigen-binding protein by injection, including bysubcutaneous injection.

In some embodiments, the method reduces the levels of one or more ofapolipoprotein B (ApoB), non-high density lipoprotein cholesterol(non-HDL-C), total cholesterol (TC), lipoprotein a (Lp(a)), high-densitylipoprotein cholesterol (HDL-C), triglyceride (TG), or ApolipoproteinA-1 (Apo A-1) in a subject.

In certain embodiments, the subject exhibits one or more symptoms orindicia of hypercholesterolemia or has been diagnosed withhypercholesterolemia, or would benefit from a reduction in total serumcholesterol, LDL, triglycerides, VLDL, lipoprotein(a), or would benefitfrom an increase in HDL.

In certain embodiments, the PCSK9-mediated disease or PCSK9-mediatedcondition is selected from the group consisting of elevated totalcholesterol levels, elevated low-density lipoprotein cholesterol (LDL-C)levels, hyperlipidemia, dyslipidemia, atherosclerosis, cardiovasculardisease, hypercholesterolemia, primary hypercholesterolemia, familialhypercholesterolemia, and hypercholesteremia which is uncontrolled bystatins. In certain embodiments, the subject falls into one or more ofthe following groups of subjects: (i) subjects having a serum LDLcholesterol (LDL-C) level of at least 100 mg/dL, (ii) subjects having aserum HDL-C level of less than 40 mg/dL; (iii) subjects having a serumcholesterol level of at least 200 mg/dL; and (iv) subjects having aserum triacylglycerol level of at least 150 mg/dL, wherein thetriacylglycerol level is determined after fasting for at least 8 hours.

The present invention also provides a pharmaceutical compositioncomprising a PCSK9 inhibitor for treating a subject with aPCSK9-mediated disease or a PCSK9-mediated condition, and apharmaceutically acceptable excipient. The PCSK9 inhibitor is anantibody in certain embodiments, including an antibody comprising aheavy chain variable domain comprising the CDR amino acid sequences setforth in SEQ ID NOs:2, 3, and 4; and a light chain variable domaincomprising the CDR amino acid sequences set forth in SEQ ID NOs:7, 8,and 10. In certain embodiments, the antibody comprises a heavy chainvariable domain comprising the CDR amino acid sequences set forth in SEQID NOs:12, 13, and 14; and a light chain variable domain comprising theCDR amino acid sequences set forth in SEQ ID NOs:16, 17, and 18. Incertain embodiments, the antibody comprises the heavy chain variabledomain and the light chain variable domain, respectively, amino acidsequences set forth in SEQ ID NOs:1 and 6 or SEQ ID NOs:11 and 15. Incertain embodiments, the pharmaceutical composition comprises apharmaceutically acceptable excipient which is a combination ofhistidine, pH 6.0, polysorbate 20, and sucrose.

One embodiment provides a dosing regimen of an anti-proproteinconvertase subtilisin/kexin type 9 (anti-PCSK9) antibody orantigen-binding protein that maintains a constant low-densitylipoprotein cholesterol (LDL-C) lowering throughout the interdosinginterval in a human subject which, following administration of theanti-PCSK9 antibody or antigen-binding protein thereof at a dose ofabout 150 mg every 4 weeks for at least 3 doses, has one or more of theproperties selected from the group consisting of: (a) an area under theplasma concentration versus time curve calculated using the trapezoidalmethod from time zero to real time (AUC_(last)) from about 250 mg·day/Lto about 650 mg·day/L; (b) a maximum plasma concentration observed(C_(max)) from about 15 mg/L to about 33 mg/L; (c) a first time to reacha maximum plasma concentration (t_(max)) of about 7 days; and (d) a timeto reach terminal half life (t_(1/2) ^(z)) from about 5.5 days to about12 days.

Another embodiment provides a dosing regimen of an anti-proproteinconvertase subtilisin/kexin type 9 (anti-PCSK9) antibody orantigen-binding protein that maintains a constant low-densitylipoprotein cholesterol (LDL-C) lowering throughout the interdosinginterval in a human subject which, following administration of theanti-PCSK9 antibody or antigen-binding protein thereof at a dose ofabout 150 mg every 4 weeks for at least 3 doses, has one or more of theproperties selected from the group consisting of: (a) an area under theplasma concentration versus time curve calculated using the trapezoidalmethod from time zero to real time (AUC_(last)) from about 150 mg·day/Lto about 450 mg·day/L; (b) a maximum plasma concentration observed(C_(max)) from about 10.5 mg/L to about 24 mg/L; (c) a first time toreach a maximum plasma concentration (t_(max)) of about 7 days; and (d)a time to reach terminal half life (t_(1/2) ^(z)) from about 5 days toabout 9 days.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph of the prior art showing the mean percent change inbaseline LDL-C versus week during treatment and follow-up period forpatients with heterozygous familial hypercholesterolaemia givenalirocumab who are on a stable statin dose with or without ezetimibetherapy. Specifically, the “saw tooth” profile of the plasma LDL-Cconcentration is evident, as the prior art treatments are not able tomaintain constant LDL-C lowering throughout the interdosing interval.

FIG. 2 is a graph showing the percent change of LDL-C from baseline fromDay −29 to Day 120 for the three groups: alirocumab+placebo,alirocumab+ezetimibe, and alirocumab+fenofibrate.

FIG. 3 is a graph showing the percent change of LDL-C from baseline fromDay −1 to Day 120 for the three groups: alirocumab+placebo,alirocumab+ezetimibe, and alirocumab+fenofibrate.

FIG. 4 is a graph showing the percent change of LDL-C from baseline plotof mean estimates of pairwise comparisons for: alirocumab 150 mg SCQ4W+ezetimibe v. alirocumab 150 mg SC Q4W; and alirocumab 150 mg SCQ4W+fenofibrate v. alirocumab 150 mg SC Q4W.

FIGS. 5A-D are a group of four graphs showing mean levels of free PCSK9,comparing the three treatment groups together (FIG. 5A) and comparedwith percent changes in LDL-C from the Day −29 baseline, per treatmentgroup (FIGS. 5B-D) (N=24 per group). FIG. 5A shows the results for allthree groups compared. FIG. 5B shows the results for thealirocumab+placebo group. FIG. 5C shows the results for thealirocumab+EZE group. FIG. 5D shows the results for the alirocumab+FENOgroup.

FIGS. 6A-C are a group of three graphs showing the percent changes inLDL-C from the Day −29 baseline (FIG. 6A), and levels of free PCSK9(FIG. 6B) and total alirocumab (FIG. 6C) from Day 57 (time of 3rdalirocumab injection) to Day 85 (28 days after 3rd alirocumabinjection).

FIG. 7 (top) are two graphs showing the mean alirocumab serumconcentration-time profiles on Day 1 after the first alirocumabadministration in linear (top left) and semi-log scale (top right) forthe three groups: alirocumab+placebo, alirocumab+ezetimibe, andalirocumab+fenofibrate. FIG. 7 (bottom) are two graphs showing meanalirocumab serum concentration-time profiles on Day 57 after the thirdalirocumab administration in linear (bottom left) and semi-log scale(bottom right) for the three groups: alirocumab+placebo,alirocumab+ezetimibe, and alirocumab+fenofibrate.

DETAILED DESCRIPTION

Before the present invention is described, it is to be understood thatthis invention is not limited to particular methods and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art.

It is noted here that as used in this specification and the appendedclaims, the singular forms “a”, “an”, and “the” also include pluralreference, unless the context clearly dictates otherwise.

The term “about” or “approximately,” when used in reference to aparticular recited numerical value, means that the value may vary fromthe recited value by no more than 1%. For example, as used herein, theexpression “about 100” includes 99 and 101 and all values in between(e.g., 99.1, 99.2, 99.3, 99.4, etc.).

The terms “administer” or “administration” refer to the act of injectingor otherwise physically delivering a substance as it exists outside thebody (e.g., a formulation of the invention) into a patient, such as bymucosal, intradermal, intravenous, subcutaneous, intramuscular deliveryand/or any other method of physical delivery described herein or knownin the art. When a disease, or a symptom thereof, is being treated,administration of the substance typically occurs after the onset of thedisease or symptoms thereof. When a disease or symptoms thereof, arebeing prevented, administration of the substance typically occurs beforethe onset of the disease or symptoms thereof.

The terms “composition” and “formulation” are intended to encompass aproduct containing the specified ingredients (e.g., an anti-PCSK9antibody) in, optionally, the specified amounts, as well as any productwhich results, directly or indirectly, from the combination of thespecified ingredients in, optionally, the specified amounts.

The term “excipients” refers to inert substances that are commonly usedas a diluent, vehicle, preservative, binder, stabilizing agent, etc. fordrugs and includes, but is not limited to, proteins (e.g., serumalbumin, etc.), amino acids (e.g., aspartic acid, glutamic acid, lysine,arginine, glycine, histidine, etc.), fatty acids and phospholipids(e.g., alkyl sulfonates, caprylate, etc.), surfactants (e.g., SDS,polysorbate, nonionic surfactant, etc.), saccharides (e.g., sucrose,maltose, trehalose, etc.) and polyols (e.g., mannitol, sorbitol, etc.).See, also, Remington's Pharmaceutical Sciences (1990) Mack PublishingCo., Easton, Pa., which is hereby incorporated by reference in itsentirety.

In the context of a peptide or polypeptide, the term “fragment” refersto a peptide or polypeptide that comprises less than the full lengthamino acid sequence. Such a fragment may arise, for example, from atruncation at the amino terminus, a truncation at the carboxy terminus,and/or an internal deletion of a residue(s) from the amino acidsequence. Fragments may, for example, result from alternative RNAsplicing or from in vivo protease activity. In certain embodiments,PCSK9 fragments include polypeptides comprising an amino acid sequenceof at least 50, at 100 amino acid residues, at least 125 contiguousamino acid residues, at least 150 contiguous amino acid residues, atleast 175 contiguous amino acid residues, at least 200 contiguous aminoacid residues, or at least 250 contiguous amino acid residues of theamino acid sequence of a PCSK9 polypeptide. In a specific embodiment, afragment of a PCSK9 polypeptide or an antibody that specifically bindsto a PCSK9 antigen retains at least 1, at least 2, or at least 3functions of the full-length polypeptide or antibody.

The term “pharmaceutically acceptable” means being approved by aregulatory agency of the Federal or a state government, or listed in theU.S. Pharmacopeia, European Pharmacopeia or other generally recognizedPharmacopeia for use in animals, and more particularly in humans.

The terms “prevent”, “preventing”, and “prevention” refer to the totalor partial inhibition of the development, recurrence, onset or spread ofa PCSK9-mediated disease and/or symptom related thereto, resulting fromthe administration of a therapy or combination of therapies providedherein (e.g., a combination of prophylactic or therapeutic agents).

The term “PCSK9 antigen” refers to that portion of a PCSK9 polypeptideto which an antibody specifically binds. A PCSK9 antigen also refers toan analog or derivative of a PCSK9 polypeptide or fragment thereof towhich an antibody specifically binds. In some embodiments, a PCSK9antigen is a monomeric PCSK9 antigen or a trimeric PCSK9 antigen. Aregion of a PCSK9 polypeptide contributing to an epitope may becontiguous amino acids of the polypeptide, or the epitope may cometogether from two or more non-contiguous regions of the polypeptide. Theepitope may or may not be a three-dimensional surface feature of theantigen. A localized region on the surface of a PCSK9 antigen that iscapable of eliciting an immune response is a PCSK9 epitope. The epitopemay or may not be a three-dimensional surface feature of the antigen.

The term “human PCSK9,” “hPCSK9” or “hPCSK9 polypeptide” and similarterms refer to the polypeptides (“polypeptides,” “peptides” and“proteins” are used interchangeably herein) comprising the amino acidsequence of SEQ ID NO:198 and related polypeptides, including SNPvariants thereof. Related polypeptides include allelic variants (e.g.,SNP variants); splice variants; fragments; derivatives; substitution,deletion, and insertion variants; fusion polypeptides; and interspecieshomologs, preferably, which retain PCSK9 activity and/or are sufficientto generate an anti-PCSK9 immune response. Also encompassed are solubleforms of PCSK9 that are sufficient to generate an anti-PCSK9immunological response. As those skilled in the art will appreciate, ananti-PCSK9 antibody can bind to a PCSK9 polypeptide, polypeptidefragment, antigen, and/or epitope, as an epitope is part of the largerantigen, which is part of the larger polypeptide fragment, which, inturn, is part of the larger polypeptide. hPCSK9 can exist in a trimeric(native) or monomeric (denatured) form.

The terms “PCSK9-mediated disease,” “PCSK9-mediated condition,” and“PCSK9-mediated disorder” are used interchangeably and refer to anydisease that is completely or partially caused by or is the result ofPCSK9, e.g., hPCSK9. In certain embodiments, PCSK9 is aberrantly (e.g.,highly) expressed. In some embodiments, PCSK9 may be aberrantlyupregulated. In other embodiments, normal, aberrant, or excessive cellsignaling is caused by binding of PCSK9 to a PCSK9 ligand. In certainembodiments, the PCSK9 ligand is a PCSK9 receptor. In certainembodiments, the PCSK9-mediated disease or condition is selected fromthe group consisting of: elevated total cholesterol levels; elevatedlow-density lipoprotein cholesterol (LDL-C) levels; hyperlipidemia;dyslipidemia; hypercholesterolemia, particularly hypercholesterolemiauncontrolled by statins, hypercholesterolemia, such as familialhypercholesterolemia or non-familial hypercholesterolemia, andhypercholesterolemia uncontrolled by statins; atherosclerosis; andcardiovascular diseases.

The terms “subject” and “patient” are used interchangeably. As usedherein, a subject is preferably a mammal, such as a non-primate (e.g.,cows, pigs, horses, cats, dogs, rats, etc.) or a primate (e.g., monkeyand human), most preferably a human. In one embodiment, the subject is amammal, preferably a human, having a PCSK9-mediated disease. In anotherembodiment, the subject is a mammal, preferably a human, at risk ofdeveloping a PCSK9-mediated disease.

The term “therapeutic agent” refers to any agent that can be used in thetreatment, management or amelioration of a PCSK9-mediated disease and/ora symptom related thereto. In certain embodiments, the term “therapeuticagent” refers to a PCSK9 antibody of the invention. In certain otherembodiments, the term “therapeutic agent” refers to an agent other thana PCSK9 antibody of the invention. Preferably, a therapeutic agent is anagent that is known to be useful for, or has been or is currently beingused for the treatment, management or amelioration of a PCSK9-mediateddisease or one or more symptoms related thereto.

The term “therapy” refers to any protocol, method, and/or agent that canbe used in the prevention, management, treatment, and/or amelioration ofa PCSK9-mediated disease (e.g., atherosclerosis orhypercholesterolemia). In certain embodiments, the terms “therapies” and“therapy” refer to a biological therapy, supportive therapy, and/orother therapies useful in the prevention, management, treatment, and/oramelioration of a PCSK9-mediated disease known to one of skill in theart, such as medical personnel.

The terms “treat”, “treatment”, and “treating” refer to the reduction oramelioration of the progression, severity, and/or duration of aPCSK9-mediated disease (e.g., atherosclerosis) resulting from theadministration of one or more therapies (including, but not limited to,the administration of one or more prophylactic or therapeutic agents).In specific embodiments, such terms refer to the reduction or inhibitionof the binding of PCSK9 to a PCSK9 ligand.

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice of the present invention,the preferred methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference to describe intheir entirety.

Patient Populations

The methods of the present invention comprise selecting subjects thathave, or are at risk of developing, a PCSK9-mediated disease orcondition, such as hypercholesterolemia or a related disorder (e.g.,atherosclerosis), and administering to these subjects, in the absence ofa statin, a pharmaceutical composition comprising a PCSK9 inhibitor.

For example, the methods of the present invention comprise administeringto a subject in need thereof a therapeutic composition comprising ananti-PCSK9 antibody, in the absence of a statin. The therapeuticcomposition can comprise any of the anti-PCSK9 antibodies, or fragmentsthereof, as disclosed herein.

As used herein, the expression “a subject in need thereof” means a humanor non-human animal that exhibits one or more symptoms or indicia ofhypercholesterolemia or who has been diagnosed withhypercholesterolemia, or who otherwise would benefit from a reduction intotal serum cholesterol, LDL, triglycerides, VLDL, lipoprotein(a)[Lp(a)], or who would benefit from an increase in HDL. Specificexemplary populations treatable by the therapeutic methods of theinvention include patients indicated for LDL apheresis, subjects withPCSK9-activating (GOF) mutations, patients with heterozygous orhomozygous Familial Hypercholesterolemia (HeFH or HoFH); subjects withprimary hypercholesterolemia who are statin intolerant or statinuncontrolled; and subjects at risk for developing hypercholesterolemiawho may be preventably treated.

While modifications in lifestyle and conventional drug treatment areoften successful in reducing cholesterol levels, not all patients areable to achieve the recommended target cholesterol levels with suchapproaches. Various conditions, such as familial hypercholesterolemia(FH), appear to be resistant to lowering of LDL-C levels in spite ofaggressive use of conventional therapy. Homozygous and heterozygousfamilial hypercholesterolemia (hoFH, heFH) are conditions associatedwith premature atherosclerotic vascular disease. However, patientsdiagnosed with hoFH are largely unresponsive to conventional drugtherapy and have limited treatment options. Specifically, treatment withstatins, which reduce LDL-C by inhibiting cholesterol synthesis andupregulating the hepatic LDL receptor, may have little effect inpatients whose LDL receptors are non-existent or defective. A mean LDL-Creduction of only less than about 20% has been recently reported inpatients with genotype-confirmed hoFH treated with the maximal dose ofstatins. The addition of ezetimibe 10 mg/day to this regimen resulted ina total reduction of LDL-C levels of 27%, which is still far fromoptimal. Likewise, many patients are statin non-responsive, poorlycontrolled with statin therapy, or cannot tolerate statin therapy; ingeneral, these patients are unable to achieve cholesterol control withalternative treatments. There is a large unmet medical need for newtreatments that can address the short-comings of current treatmentoptions.

Thus, the invention includes therapeutic methods in which a PCSK9inhibitor of the invention is administered in the absence of a statin toa patient to treat or prevent hypercholesterolemia. As used herein, theuse of the PCSK9 inhibitor “in the absence of a statin” means that thesubject is not taking a concomitant statin while being treated with thePCSK9 inhibitor of the invention, or was not recently taking a statinprior to treatment with the PCSK9 inhibitor of the invention. The terms“in the absence of a statin” and “not on a concomitant statin” mean thatthe subject should have no detectable levels of statin the bloodstream,but, due to prior therapy, the subject may have a serum concentration ofany statin of less than 0.1 mg/mL. As used herein, a “lipid loweringagent” means any pharmaceutical agent other than a PCSK9 inhibitor whichis administered for the purpose of modifying the lipid profile of asubject. Examples of lipid-lowering agents include, but are not limitedto: HMG-CoA reductase inhibitors, including statins (atorvastatin,cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin,pravastatin, rosuvastatin, simvastatin, etc.), niacin, fibric acid, bileacid sequestrants (e.g., cholestyramine), colesevelam, colestipol, andezetimibe. It follows that a “non-statin lipid lowering agent” means anypharmaceutical agent other than a PCSK9 inhibitor and a statin. Examplesof non-statin lipid lowering agents include, but are not limited to,niacin, fibric acid, fenofibrate, bile acid sequestrants (e.g.,cholestyramine), colesevelam, colestipol, an omega-3 fatty acid, a bileacid resin, and ezetimibe.

In some instances the patient who is treated with a therapeuticformulation of the present invention is otherwise healthy except forexhibiting elevated levels of cholesterol, lipids, triglycerides orlipoproteins. For example, the patient may not exhibit any other riskfactor of cardiovascular, thrombotic or other diseases or disorders atthe time of treatment. In other instances, however, the patient isselected on the basis of being diagnosed with, or at risk of developing,a disease or disorder that is caused by, correlated with or ancillary toelevated serum cholesterol, lipids, triglycerides or lipoproteins. Forexample, at the time of, or prior to administration of thepharmaceutical composition of the present invention, the patient may bediagnosed with or identified as being at risk of developing acardiovascular disease or disorder, such as, e.g., coronary arterydisease, acute myocardial infarction, asymptomatic carotidatherosclerosis, stroke, peripheral artery occlusive disease, etc. Thecardiovascular disease or disorder, in some instances, ishypercholesterolemia. For example, a patient may be selected fortreatment with a pharmaceutical composition of the present invention ifthe patient is diagnosed with or identified as being at risk ofdeveloping a hypercholesterolemia condition such as, e.g., heterozygousFamilial Hypercholesterolemia (heFH), homozygous FamilialHypercholesterolemia (hoFH), as well as incidences ofhypercholesterolemia that are distinct from FamilialHypercholesterolemia (nonFH).

In other instances, at the time of, or prior to administration of thepharmaceutical composition of the present invention, the patient may bediagnosed with or identified as being at risk of developing a thromboticocclusive disease or disorder, such as, e.g., pulmonary embolism,central retinal vein occlusion, etc. In certain embodiments, the patientis selected on the basis of being diagnosed with or at risk ofdeveloping a combination of two or more of the above mentioned diseasesor disorders. For example, at the time of, or prior to administration ofthe pharmaceutical composition of the present invention, the patient maybe diagnosed with or identified as being at risk of developing coronaryartery disease and pulmonary embolism. Other diagnostic combinations(e.g., atherosclerosis and central retinal vein occlusion, heFH andstroke, etc.) are also included in the definition of the patientpopulations that are treatable with a pharmaceutical composition of thepresent invention.

The pharmaceutical compositions of the present invention are also usefulfor treating hypercholesterolemia or dyslipidemia caused by or relatedto an underlying disease or disorder selected from the group consistingof metabolic syndrome, diabetes mellitus, hypothyroidism, nephroticsyndrome, renal failure, Cushing's syndrome, biliary cirrhosis, glycogenstorage diseases, hepatoma, cholestasis, growth hormone deficiency. Thepharmaceutical compositions of the present invention are also useful fortreating hypercholesterolemia or dyslipidemia caused by or related to aprior therapeutic regimen such as estrogen therapy, progestin therapy,beta-blockers, or diuretics.

In yet other instances, the patient who is to be treated with apharmaceutical composition of the present invention is selected on thebasis of one or more factors selected from the group consisting of age(e.g., older than 40, 45, 50, 55, 60, 65, 70, 75, or 80 years), race,gender (male or female), exercise habits (e.g., regular exerciser,non-exerciser), other preexisting medical conditions (e.g., type-IIdiabetes, high blood pressure, etc.), and current medication status(e.g., currently taking statins [e.g., cerivastatin, atorvastatin,simvastatin, pitavastatin, rosuvastatin, fluvastatin, lovastatin,pravastatin, etc.], beta blockers, niacin, etc.). Potential patients canbe selected/screened on the basis of one or more of these factors (e.g.,by questionnaire, diagnostic evaluation, etc.) before being treated withthe methods of the present invention.

The present invention also includes methods for increasingtransintestinal cholesterol excretion (TICE) in a subject byadministering a PCSK9 inhibitor to the subject. For example, the presentinvention provides methods for increasing TICE in a subject byadministering to the subject an anti-PCSK9 antibody with pH-dependentbinding characteristics. According to certain embodiments, the presentinvention includes methods comprising identifying a subject for whichenhanced TICE would be beneficial, or identifying a subject thatexhibits impaired TICE, and administering a PCSK9 inhibitor to thesubject.

Hypercholesterolemia is a precursor to atherosclerosis. Accordingly, theinvention also includes therapeutic methods in which a PCSK9 inhibitorof the invention is administered in the absence of a statin to a patientto treat or prevent atherosclerosis. Risk factors for atherosclerosisare well known in the art and include, without limitation, high lowdensity lipoprotein (LDL) cholesterol levels, low high densitylipoprotein (HDL) cholesterol levels, hypertension, diabetes mellitus,family history, male gender, cigarette smoking, and high serumcholesterol. Methods of assessing these risk factors for a given subjectare also well known in the art.

In certain embodiments, the selected subject is hyperlipidemic. A“hyperlipidemic” is a subject that is a hypercholesterolemic and/or ahypertriglyceridemic subject. A “hypercholesterolemic” subject is onethat fits the current criteria established for a hypercholesterolemicsubject. A “hypertriglyceridemic” subject is one that fits the currentcriteria established for a hypertriglyceridemic subject (See, e.g.,Harrison's Principles of Experimental Medicine, 13th Edition,McGraw-Hill, Inc., N.Y). For example, a hypercholesterolemic subjecttypically has an LDL level of >160 mg/dL, or >130 mg/dL and at least tworisk factors selected from the group consisting of male gender, familyhistory of premature coronary heart disease, cigarette smoking (morethan 10 per day), hypertension, low HDL (<35 mg/dL), diabetes mellitus,hyperinsulinemia, abdominal obesity, high lipoprotein (a), and personalhistory of cerebrovascular disease or occlusive peripheral vasculardisease. A hypertriglyceridemic subject typically has a triglyceride(TG) level of >250 mg/dL. In certain embodiments the selected subject ishyperlipidemic but not receiving treatment for hyperlipidemia.

PCSK9 Inhibitors

The methods of the present invention comprise administering to a patienta therapeutic composition comprising a PCSK9 inhibitor. As used herein,a “PCSK9 inhibitor” is any agent which binds to or interacts with humanPCSK9 and inhibits the normal biological function of PCSK9 in vitro orin vivo. Non-limiting examples of categories of PCSK9 inhibitors includesmall molecule PCSK9 antagonists, peptide-based PCSK9 antagonists (e.g.,“peptibody” molecules), and antibodies or antigen-binding fragments ofantibodies that specifically bind human PCSK9.

The term “human proprotein convertase subtilisin/kexin type 9” or “humanPCSK9” or “hPCSK9” refers to PCSK9 having the nucleic acid sequenceshown in SEQ ID NO:197 and the amino acid sequence of SEQ ID NO:198, ora biologically active fragment thereof.

The term “antigen binding protein” means a protein that binds to anantigen. For example, an antigen binding protein includes, but is notlimited to, an antibody, an antigen binding fragment of an antibody, aDVD-Ig, and a dual variable domain immunoglobulin.

The term “antibody” is intended to refer to immunoglobulin moleculescomprising four polypeptide chains, two heavy (H) chains and two light(L) chains inter-connected by disulfide bonds, as well as multimersthereof (e.g., IgM). Each heavy chain comprises a heavy chain variableregion (abbreviated herein as HCVR or V_(H)) and a heavy chain constantregion. The heavy chain constant region comprises three domains, C_(H)1,C_(H)2 and C_(H)3. Each light chain comprises a light chain variableregion (abbreviated herein as LCVR or V_(L)) and a light chain constantregion. The light chain constant region comprises one domain (C_(L)1).The V_(H) and V_(L) regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDRs),interspersed with regions that are more conserved, termed frameworkregions (FR). Each V_(H) and V_(L) is composed of three CDRs and fourFRs, arranged from amino-terminus to carboxy-terminus in the followingorder: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments ofthe invention, the FRs of the anti-PCSK9 antibody (or antigen-bindingportion thereof) may be identical to the human germline sequences, ormay be naturally or artificially modified. An amino acid consensussequence may be defined based on a side-by-side analysis of two or moreCDRs.

The term “antibody,” also includes antigen-binding fragments of fullantibody molecules. The terms “antigen-binding portion” of an antibody,“antigen-binding fragment” of an antibody, and the like, include anynaturally occurring, enzymatically obtainable, synthetic, or geneticallyengineered polypeptide or glycoprotein that specifically binds anantigen to form a complex. Antigen-binding fragments of an antibody maybe derived, e.g., from full antibody molecules using any suitablestandard techniques such as proteolytic digestion or recombinant geneticengineering techniques involving the manipulation and expression of DNAencoding antibody variable and optionally constant domains. Such DNA isknown and/or is readily available from, e.g., commercial sources, DNAlibraries (including, e.g., phage-antibody libraries), or can besynthesized. The DNA may be sequenced and manipulated chemically or byusing molecular biology techniques, for example, to arrange one or morevariable and/or constant domains into a suitable configuration, or tointroduce codons, create cysteine residues, modify, add or delete aminoacids, etc.

Non-limiting examples of antigen-binding fragments include: (i) Fabfragments; (ii) F(ab′)2 fragments; (iii) Fd fragments; (iv) Fvfragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and(vii) minimal recognition units consisting of the amino acid residuesthat mimic the hypervariable region of an antibody (e.g., an isolatedcomplementarity determining region (CDR) such as a CDR3 peptide), or aconstrained FR3-CDR3-FR4 peptide. Other engineered molecules, such asdomain-specific antibodies, single domain antibodies, domain-deletedantibodies, chimeric antibodies, CDR-grafted antibodies, diabodies,triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalentnanobodies, bivalent nanobodies, etc.), small modularimmunopharmaceuticals (SMIPs), and shark variable IgNAR domains, arealso encompassed within the expression “antigen-binding fragment”.

An antigen-binding fragment of an antibody will typically comprise atleast one variable domain. The variable domain may be of any size oramino acid composition and will generally comprise at least one CDRwhich is adjacent to or in frame with one or more framework sequences.In antigen-binding fragments having a V_(H) domain associated with aV_(L) domain, the V_(H) and V_(L) domains may be situated relative toone another in any suitable arrangement. For example, the variableregion may be dimeric and contain V_(H)-V_(H), V_(H)-V_(L) orV_(L)-V_(L) dimers. Alternatively, the antigen-binding fragment of anantibody may contain a monomeric V_(H) or V_(L) domain.

In certain embodiments, an antigen-binding fragment of an antibody maycontain at least one variable domain covalently linked to at least oneconstant domain. Non-limiting, exemplary configurations of variable andconstant domains that may be found within an antigen-binding fragment ofan antibody of the present invention include: (i) V_(H)-C_(H)1; (ii)V_(H)-C_(H)2; (iii) V_(H)-C_(H)3; (iv) V_(H)-C_(H)1-C_(H)2; (v)V_(H)-C_(H)1-C_(H)2-C_(H)3; (vi) V_(H)-C_(H)2-C_(H)3; (vii) V_(H)-C_(L);(viii) V_(L)-C_(H)1; (ix) V_(L)-C_(H)2; (x) V_(L)-C_(H)3; (xi)V_(L)-C_(H)1-C_(H)2; (xii) V_(L)-C_(H)1-C_(H)2-C_(H)3; (xiii)V_(L)-C_(H)2-C_(H)3; and (xiv) V_(L)-C_(L). In any configuration ofvariable and constant domains, including any of the exemplaryconfigurations listed above, the variable and constant domains may beeither directly linked to one another or may be linked by a full orpartial hinge or linker region. A hinge region may consist of at least 2(e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in aflexible or semi-flexible linkage between adjacent variable and/orconstant domains in a single polypeptide molecule. Moreover, anantigen-binding fragment of an antibody of the present invention maycomprise a homo-dimer or hetero-dimer (or other multimer) of any of thevariable and constant domain configurations listed above in non-covalentassociation with one another and/or with one or more monomeric V_(H) orV_(L) domain (e.g., by disulfide bond(s)).

As with full antibody molecules, antigen-binding fragments may bemonospecific or multispecific (e.g., bispecific). A multispecificantigen-binding fragment of an antibody will typically comprise at leasttwo different variable domains, wherein each variable domain is capableof specifically binding to a separate antigen or to a different epitopeon the same antigen. Any multispecific antibody format, including theexemplary bispecific antibody formats disclosed herein, may be adaptedfor use in the context of an antigen-binding fragment of an antibody ofthe present invention using routine techniques available in the art.

The constant region of an antibody is important in the ability of anantibody to fix complement and mediate cell-dependent cytotoxicity.Thus, the isotype of an antibody may be selected on the basis of whetherit is desirable for the antibody to mediate cytotoxicity.

The term “human antibody” is intended to include antibodies havingvariable and constant regions derived from human germline immunoglobulinsequences. The human antibodies of the invention may nonetheless includeamino acid residues not encoded by human germline immunoglobulinsequences (e.g., mutations introduced by random or site-specificmutagenesis in vitro or by somatic mutation in vivo), for example in theCDRs and in particular CDR3. However, the term “human antibody” is notintended to include antibodies in which CDR sequences derived from thegermline of another mammalian species, such as a mouse, have beengrafted onto human framework sequences.

The term “recombinant human antibody” is intended to include all humanantibodies that are prepared, expressed, created or isolated byrecombinant means, such as antibodies expressed using a recombinantexpression vector transfected into a host cell (described furtherbelow), antibodies isolated from a recombinant, combinatorial humanantibody library (described further below), antibodies isolated from ananimal (e.g., a mouse) that is transgenic for human immunoglobulin genes(see e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295) orantibodies prepared, expressed, created or isolated by any other meansthat involves splicing of human immunoglobulin gene sequences to otherDNA sequences. Such recombinant human antibodies have variable andconstant regions derived from human germline immunoglobulin sequences.In certain embodiments, however, such recombinant human antibodies aresubjected to in vitro mutagenesis (or, when an animal transgenic forhuman Ig sequences is used, in vivo somatic mutagenesis) and thus theamino acid sequences of the V_(H) and V_(L) regions of the recombinantantibodies are sequences that, while derived from and related to humangermline V_(H) and V_(L) sequences, may not naturally exist within thehuman antibody germline repertoire in vivo.

Human antibodies can exist in two forms that are associated with hingeheterogeneity. In one form, an immunoglobulin molecule comprises astable four chain construct of approximately 150-160 kDa in which thedimers are held together by an interchain heavy chain disulfide bond. Ina second form, the dimers are not linked via inter-chain disulfide bondsand a molecule of about 75-80 kDa is formed composed of a covalentlycoupled light and heavy chain (half-antibody). These forms have beenextremely difficult to separate, even after affinity purification.

The frequency of appearance of the second form in various intact IgGisotypes is due to, but not limited to, structural differencesassociated with the hinge region isotype of the antibody. A single aminoacid substitution in the hinge region of the human IgG4 hinge cansignificantly reduce the appearance of the second form (Angal et al.(1993) Molecular Immunology 30:105) to levels typically observed using ahuman IgG1 hinge. The instant invention encompasses antibodies havingone or more mutations in the hinge, C_(H)2 or C_(H)3 region which may bedesirable, for example, in production, to improve the yield of thedesired antibody form.

An “isolated antibody” means an antibody that has been identified andseparated and/or recovered from at least one component of its naturalenvironment. For example, an antibody that has been separated or removedfrom at least one component of an organism, or from a tissue or cell inwhich the antibody naturally exists or is naturally produced, is an“isolated antibody” for purposes of the present invention. An isolatedantibody also includes an antibody in situ within a recombinant cell.Isolated antibodies are antibodies that have been subjected to at leastone purification or isolation step. According to certain embodiments, anisolated antibody may be substantially free of other cellular materialand/or chemicals.

The term “specifically binds” or the like, means that an antibody orantigen-binding fragment thereof forms a complex with an antigen that isrelatively stable under physiologic conditions. Methods for determiningwhether an antibody specifically binds to an antigen are well known inthe art and include, for example, equilibrium dialysis, surface plasmonresonance, and the like. For example, an antibody that “specificallybinds” PCSK9, as used in the context of the present invention, includesantibodies that bind PCSK9 or portion thereof with a K_(D) of less thanabout 1000 nM, less than about 500 nM, less than about 300 nM, less thanabout 200 nM, less than about 100 nM, less than about 90 nM, less thanabout 80 nM, less than about 70 nM, less than about 60 nM, less thanabout 50 nM, less than about 40 nM, less than about 30 nM, less thanabout 20 nM, less than about 10 nM, less than about 5 nM, less thanabout 4 nM, less than about 3 nM, less than about 2 nM, less than about1 nM or less than about 0.5 nM, as measured in a surface plasmonresonance assay. An isolated antibody that specifically binds humanPCSK9, however, have cross-reactivity to other antigens, such as PCSK9molecules from other (non-human) species.

The anti-PCSK9 antibodies useful for the methods of the presentinvention may comprise one or more amino acid substitutions, insertionsand/or deletions in the framework and/or CDR regions of the heavy andlight chain variable domains as compared to the corresponding germlinesequences from which the antibodies were derived. Such mutations can bereadily ascertained by comparing the amino acid sequences disclosedherein to germline sequences available from, for example, publicantibody sequence databases. The present invention includes methodsinvolving the use of antibodies, and antigen-binding fragments thereof,which are derived from any of the amino acid sequences disclosed herein,wherein one or more amino acids within one or more framework and/or CDRregions are mutated to the corresponding residue(s) of the germlinesequence from which the antibody was derived, or to the correspondingresidue(s) of another human germline sequence, or to a conservativeamino acid substitution of the corresponding germline residue(s) (suchsequence changes are referred to herein collectively as “germlinemutations”). A person of ordinary skill in the art, starting with theheavy and light chain variable region sequences disclosed herein, caneasily produce numerous antibodies and antigen-binding fragments whichcomprise one or more individual germline mutations or combinationsthereof. In certain embodiments, all of the framework and/or CDRresidues within the V_(H) and/or V_(L) domains are mutated back to theresidues found in the original germline sequence from which the antibodywas derived. In other embodiments, only certain residues are mutatedback to the original germline sequence, e.g., only the mutated residuesfound within the first 8 amino acids of FR1 or within the last 8 aminoacids of FR4, or only the mutated residues found within CDR1, CDR2 orCDR3. In other embodiments, one or more of the framework and/or CDRresidue(s) are mutated to the corresponding residue(s) of a differentgermline sequence (i.e., a germline sequence that is different from thegermline sequence from which the antibody was originally derived).Furthermore, the antibodies of the present invention may contain anycombination of two or more germline mutations within the frameworkand/or CDR regions, e.g., wherein certain individual residues aremutated to the corresponding residue of a particular germline sequencewhile certain other residues that differ from the original germlinesequence are maintained or are mutated to the corresponding residue of adifferent germline sequence. Once obtained, antibodies andantigen-binding fragments that contain one or more germline mutationscan be easily tested for one or more desired property such as, improvedbinding specificity, increased binding affinity, improved or enhancedantagonistic or agonistic biological properties (as the case may be),reduced immunogenicity, etc. The use of antibodies and antigen-bindingfragments obtained in this general manner are encompassed within thepresent invention.

The present invention also includes methods involving the use ofanti-PCSK9 antibodies comprising variants of any of the HCVR, LCVR,and/or CDR amino acid sequences disclosed herein having one or moreconservative substitutions. For example, the present invention includesthe use of anti-PCSK9 antibodies having HCVR, LCVR, and/or CDR aminoacid sequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 orfewer, etc. conservative amino acid substitutions relative to any of theHCVR, LCVR, and/or CDR amino acid sequences disclosed herein.

The term “surface plasmon resonance” refers to an optical phenomenonthat allows for the analysis of real-time interactions by detection ofalterations in protein concentrations within a biosensor matrix, forexample using the BIAcore™ system (Biacore Life Sciences division of GEHealthcare, Piscataway, NJ).

The term “K_(D)” is intended to refer to the equilibrium dissociationconstant of a particular antibody-antigen interaction.

The term “epitope” refers to an antigenic determinant that interactswith a specific antigen binding site in the variable region of anantibody molecule known as a paratope. A single antigen may have morethan one epitope. Thus, different antibodies may bind to different areason an antigen and may have different biological effects. Epitopes may beeither conformational or linear. A conformational epitope is produced byspatially juxtaposed amino acids from different segments of the linearpolypeptide chain. A linear epitope is one produced by adjacent aminoacid residues in a polypeptide chain. In certain circumstance, anepitope may include moieties of saccharides, phosphoryl groups, orsulfonyl groups on the antigen.

According to certain embodiments, the anti-PCSK9 antibody used in themethods of the present invention is an antibody with pH-dependentbinding characteristics. As used herein, the expression “pH-dependentbinding” means that the antibody or antigen-binding fragment thereofexhibits “reduced binding to PCSK9 at acidic pH as compared to neutralpH” (for purposes of the present disclosure, both expressions may beused interchangeably). For the example, antibodies “with pH-dependentbinding characteristics” includes antibodies and antigen-bindingfragments thereof that bind PCSK9 with higher affinity at neutral pHthan at acidic pH. In certain embodiments, the antibodies andantigen-binding fragments of the present invention bind PCSK9 with atleast 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, 100, or more times higher affinity at neutral pH than atacidic pH.

According to this aspect of the invention, the anti-PCSK9 antibodieswith pH-dependent binding characteristics may possess one or more aminoacid variations relative to the parental anti-PCSK9 antibody. Forexample, an anti-PCSK9 antibody with pH-dependent bindingcharacteristics may contain one or more histidine substitutions orinsertions, e.g., in one or more CDRs of a parental anti-PCSK9 antibody.Thus, according to certain embodiments of the present invention, methodsare provided comprising administering an anti-PCSK9 antibody whichcomprises CDR amino acid sequences (e.g., heavy and light chain CDRs)which are identical to the CDR amino acid sequences of a parentalanti-PCSK9 antibody, except for the substitution of one or more aminoacids of one or more CDRs of the parental antibody with a histidineresidue. The anti-PCSK9 antibodies with pH-dependent binding maypossess, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or more histidinesubstitutions, either within a single CDR of a parental antibody ordistributed throughout multiple (e.g., 2, 3, 4, 5, or 6) CDRs of aparental anti-PCSK9 antibody. For example, the present inventionincludes the use of anti-PCSK9 antibodies with pH-dependent bindingcomprising one or more histidine substitutions in HCDR1, one or morehistidine substitutions in HCDR2, one or more histidine substitutions inHCDR3, one or more histidine substitutions in LCDR1, one or morehistidine substitutions in LCDR2, and/or one or more histidinesubstitutions in LCDR3, of a parental anti-PCSK9 antibody.

As used herein, the expression “acidic pH” means a pH of 6.0 or less(e.g., less than about 6.0, less than about 5.5, less than about 5.0,etc.). The expression “acidic pH” includes pH values of about 6.0, 5.95,5.90, 5.85, 5.8, 5.75, 5.7, 5.65, 5.6, 5.55, 5.5, 5.45, 5.4, 5.35, 5.3,5.25, 5.2, 5.15, 5.1, 5.05, 5.0, or less. As used herein, the expression“neutral pH” means a pH of about 7.0 to about 7.4. The expression“neutral pH” includes pH values of about 7.0, 7.05, 7.1, 7.15, 7.2,7.25, 7.3, 7.35, and 7.4.

Preparation of Human Antibodies

Methods for generating human antibodies in transgenic mice are known inthe art. Any such known methods can be used in the context of thepresent invention to make human antibodies that specifically bind tohuman PCSK9.

Using VELOCIMMUNE™ technology (see, for example, U.S. Pat. No.6,596,541, Regeneron Pharmaceuticals) or any other known method forgenerating monoclonal antibodies, high affinity chimeric antibodies toPCSK9 are initially isolated having a human variable region and a mouseconstant region. The VELOCIMMUNE® technology involves generation of atransgenic mouse having a genome comprising human heavy and light chainvariable regions operably linked to endogenous mouse constant regionloci such that the mouse produces an antibody comprising a humanvariable region and a mouse constant region in response to antigenicstimulation. The DNA encoding the variable regions of the heavy andlight chains of the antibody are isolated and operably linked to DNAencoding the human heavy and light chain constant regions. The DNA isthen expressed in a cell capable of expressing the fully human antibody.

Generally, a VELOCIMMUNE® mouse is challenged with the antigen ofinterest, and lymphatic cells (such as B-cells) are recovered from themice that express antibodies. The lymphatic cells may be fused with amyeloma cell line to prepare immortal hybridoma cell lines, and suchhybridoma cell lines are screened and selected to identify hybridomacell lines that produce antibodies specific to the antigen of interest.DNA encoding the variable regions of the heavy chain and light chain maybe isolated and linked to desirable isotypic constant regions of theheavy chain and light chain. Such an antibody protein may be produced ina cell, such as a CHO cell. Alternatively, DNA encoding theantigen-specific chimeric antibodies or the variable domains of thelight and heavy chains may be isolated directly from antigen-specificlymphocytes.

Initially, high affinity chimeric antibodies are isolated having a humanvariable region and a mouse constant region. The antibodies arecharacterized and selected for desirable characteristics, includingaffinity, selectivity, epitope, etc, using standard procedures known tothose skilled in the art. The mouse constant regions are replaced with adesired human constant region to generate the fully human antibody ofthe invention, for example wild-type or modified IgG1 or IgG4. While theconstant region selected may vary according to specific use, highaffinity antigen-binding and target specificity characteristics residein the variable region.

In general, the antibodies that can be used in the methods of thepresent invention possess high affinities, as described above, whenmeasured by binding to antigen either immobilized on solid phase or insolution phase. The mouse constant regions are replaced with desiredhuman constant regions to generate the fully human antibodies of theinvention. While the constant region selected may vary according tospecific use, high affinity antigen-binding and target specificitycharacteristics reside in the variable region.

Specific examples of human antibodies or antigen-binding fragments ofantibodies that specifically bind PCSK9 which can be used in the contextof the methods of the present invention include any antibody orantigen-binding fragment which comprises the three heavy chain CDRs(HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region(HCVR) having an amino acid sequence selected from the group consistingof SEQ ID NOs:1 and 11, or a substantially similar sequence thereofhaving at least 90%, at least 95%, at least 98% or at least 99% sequenceidentity. The antibody or antigen-binding fragment may comprise thethree light chain CDRs (LCVR1, LCVR2, LCVR3) contained within a lightchain variable region (LCVR) having an amino acid sequence selected fromthe group consisting of SEQ ID NOs:6 and 15, or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity.

In certain embodiments of the present invention, the antibody orantigen-binding fragment thereof comprises the six CDRs (HCDR1, HCDR2,HCDR3, LCDR1, LCDR2 and LCDR3) from the heavy and light chain variableregion amino acid sequence pairs (HCVR/LCVR) selected from the groupconsisting of SEQ ID NOs: 1/6 and 11/15.

In certain embodiments of the present invention, the anti-PCSK9antibody, or antigen-binding fragment thereof, that can be used in themethods of the present invention has HCDR1/HCDR2/HCDR3/LCDR1/LCDR2/LCDR3amino acid sequences selected from SEQ ID NOs:2/3/4/7/8/10 (mAb316P) andSEQ ID NOs:12/13/14/16/17/18 (mAb300N) (See US Patent App. Publ No.2010/0166768).

In certain embodiments of the present invention, the antibody orantigen-binding fragment thereof comprises HCVR/LCVR amino acid sequencepairs selected from the group consisting of SEQ ID NOs: 1/6 and 11/15.

Pharmaceutical Compositions and Methods of Administration

The present invention includes methods which comprise administering aPCSK9 inhibitor to a subject in the absence of a statin, wherein thePCSK9 inhibitor is contained within a pharmaceutical composition. Thepharmaceutical compositions of the invention are formulated withsuitable carriers, excipients, and other agents that provide suitabletransfer, delivery, tolerance, and the like. A multitude of appropriateformulations can be found in the formulary known to all pharmaceuticalchemists: Remington's Pharmaceutical Sciences, Mack Publishing Company,Easton, PA. These formulations include, for example, powders, pastes,ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)containing vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrousabsorption pastes, oil-in-water and water-in-oil emulsions, emulsionscarbowax (polyethylene glycols of various molecular weights), semi-solidgels, and semi-solid mixtures containing carbowax. See also Powell etal. “Compendium of excipients for parenteral formulations” PDA (1998) JPharm Sci Technol 52:238-311.

Various delivery systems are known and can be used to administer thepharmaceutical composition of the invention, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the mutant viruses, receptor mediated endocytosis (see, e.g.,Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods ofadministration include, but are not limited to, intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, and oral routes. The composition may be administered by anyconvenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents.

A pharmaceutical composition of the present invention can be deliveredsubcutaneously or intravenously with a standard needle and syringe. Inaddition, with respect to subcutaneous delivery, a pen delivery devicereadily has applications in delivering a pharmaceutical composition ofthe present invention. Such a pen delivery device can be reusable ordisposable. A reusable pen delivery device generally utilizes areplaceable cartridge that contains a pharmaceutical composition. Onceall of the pharmaceutical composition within the cartridge has beenadministered and the cartridge is empty, the empty cartridge can readilybe discarded and replaced with a new cartridge that contains thepharmaceutical composition. The pen delivery device can then be reused.In a disposable pen delivery device, there is no replaceable cartridge.Rather, the disposable pen delivery device comes prefilled with thepharmaceutical composition held in a reservoir within the device. Oncethe reservoir is emptied of the pharmaceutical composition, the entiredevice is discarded.

Numerous reusable pen and autoinjector delivery devices haveapplications in the subcutaneous delivery of a pharmaceuticalcomposition of the present invention. Examples include, but are notlimited to, AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX75/25¹m pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly and Co.,Indianapolis, IN), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen,Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen(Becton Dickinson, Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPENSTARLET™, and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany), to nameonly a few. Examples of disposable pen delivery devices havingapplications in subcutaneous delivery of a pharmaceutical composition ofthe present invention include, but are not limited to, the SOLOSTAR™ pen(sanofi-aventis), the FLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (EliLilly), the SURECLICK™ Autoinjector (Amgen, Thousand Oaks, CA), thePENLET™ (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L.P.), andthe HUMIRA™ Pen (Abbott Labs, Abbott Park Ill.), to name only a few.

In certain embodiments, the pharmaceutical composition is delivered in acontrolled release system. In certain embodiments, a pump may be used(see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201).In another embodiment, polymeric materials can be used; see, MedicalApplications of Controlled Release, Langer and Wise (eds.), 1974, CRCPres., Boca Raton, Florida. In yet another embodiment, a controlledrelease system can be placed in proximity of the composition's target,thus requiring only a fraction of the systemic dose (see, e.g., Goodson,1984, in Medical Applications of Controlled Release, supra, vol. 2, pp.115-138). Other controlled release systems are discussed in the reviewby Langer, 1990, Science 249:1527-1533.

The injectable preparations may include dosage forms for intravenous,subcutaneous, intracutaneous and intramuscular injections, dripinfusions, etc. These injectable preparations may be prepared by knownmethods. For example, the injectable preparations may be prepared, e.g.,by dissolving, suspending or emulsifying the antibody or its saltdescribed above in a sterile aqueous medium or an oily mediumconventionally used for injections. As the aqueous medium forinjections, there are, for example, physiological saline, an isotonicsolution containing glucose and other auxiliary agents, etc., which maybe used in combination with an appropriate solubilizing agent such as analcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol,polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80,HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil],etc. As the oily medium, there are employed, e.g., sesame oil, soybeanoil, etc., which may be used in combination with a solubilizing agentsuch as benzyl benzoate, benzyl alcohol, etc. In certain embodiments,the injection thus prepared is filled in an appropriate ampoule. Incertain embodiments, the pharmaceutically acceptable excipient is acombination of histidine, pH 6.0, polysorbate 20, and sucrose.

Advantageously, the pharmaceutical compositions for oral or parenteraluse described above are prepared into dosage forms in a unit dose suitedto fit a dose of the active ingredients. Such dosage forms in a unitdose include, for example, tablets, pills, capsules, injections(ampoules), suppositories, etc.

Dosage and Administration Regimens

The amount of PCSK9 inhibitor (e.g., anti-PCSK9 antibody) administeredto a subject according to the methods and compositions of the presentinvention is, generally, a therapeutically effective amount. The phrase“therapeutically effective amount” means a dose of PCSK9 inhibitor thatresults in a detectable reduction in one more symptoms ofhypercholesterolemia or a related disorder (e.g., lipid levels and/oratherosclerotic lesions).

The amount of anti-PCSK9 antibody contained within the individual dosesmay be expressed in terms of milligrams of antibody per kilogram ofpatient body weight (i.e., mg/kg). For example, the anti-PCSK9 antibodymay be administered to a patient at a dose of about 0.0001 to about 10mg/kg of patient body weight. Exemplary therapeutically effectiveamounts of antibody can be from about 75 mg, about 80 mg, about 90 mg,about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg,about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg,about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg,about 250 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg,about 300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg,about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg,about 400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg,about 450 mg, about 460 mg, about 470 mg, about 480 mg, about 490 mg,about 500 mg, about 510 mg, about 520 mg, about 530 mg, about 540 mg,about 550 mg, about 560 mg, about 570 mg, about 580 mg, about 590 mg, orabout 600 mg, of the anti-PCSK9 antibody.

In certain embodiments, the anti-PCSK9 antibody is administered to asubject at a dose of about 150 mg every four weeks for at least threedoses.

In some embodiments, the antibody is administered to a subject at a doseof about 150 mg every four weeks for 12 weeks, and the dose remains at150 mg every four weeks for another 12 weeks if, at week 8, thesubject's LDL-C value was less than 100 mg/dl and a 30% reduction ofLDL-C.

In other embodiments, the antibody is administered to a subject at adose of about 150 mg every four weeks for 12 weeks, and the dose istitrated up to about 150 mg every two weeks for another 12 weeks if, atweek 8, the subject's LDL-C value was greater than or equal to 100mg/dl.

In some embodiments, the antibody is administered to a subject at a doseof about 150 mg every four weeks for 12 weeks, and the dose remains at150 mg every four weeks for another 12 weeks if, at week 8, thesubject's LDL-C value was less than mg/dl and a 30% reduction of LDL-C.

In another embodiment, the antibody is administered to a subject at adose of about 300 mg every four weeks for 48 weeks.

In a further embodiment, the antibody is administered to a subject at adose of about 300 mg every four weeks for a total of three doses, andthe dose is changed to 150 mg every two weeks for another 36 weeks if,at week 8, the subject did not achieve a pre-determined treatment goalor the subject did not have at least a 30% reduction of LDL-C frombaseline.

Additional Therapies

In some embodiments, the invention relates to a method for increasingthe duration of action of a proprotein convertase subtilisin/kexin type9 (PCSK9) antagonist in a subject comprising administering to thesubject an anti-PCSK9 antagonist in the absence of a statin. In someembodiments, antagonist is an antibody or antigen binding protein. Forexample, the Examples show that administering an anti-PCSK9 antibody toa subject in the absence of a statin increases the duration of action ofthe anti-PCSK9 antibody.

In some embodiments, the invention relates to a method for maintainingconstant low-density lipoprotein cholesterol (LDL-C) lowering throughoutan interdosing interval in a subject comprising administering to thesubject, who is not taking a concomitant statin, a pharmaceuticalcomposition comprising an anti-proprotein convertase subtilisin/kexintype 9 (anti-PCSK9) antibody or antigen-binding protein at a dose ofabout 150 mg every 4 weeks for at least 3 doses. As shown in FIG. 1 ,prior art therapies exhibit a “sawtooth profile” of LDL-C duringtreatment. In contrast, the Q4W dosing regimen maintains constant LDL-Clowering throughout the interdosing interval in patients not receiving astatin.

In some embodiments, the invention relates to a method for reducinglow-density lipoprotein cholesterol (LDL-C) in a subject in need thereofcomprising administering to the subject, who is not taking a concomitantstatin, a pharmaceutical composition comprising an anti-proproteinconvertase subtilisin/kexin type 9 (anti-PCSK9) antibody orantigen-binding protein at a dose of about 150 mg every 4 weeks for atleast 3 doses, thereby reducing the LDL-C in the subject.

In some embodiments, the invention relates to a method for treatinghypercholesterolemia in a subject in need thereof comprisingadministering to the subject, who is not taking a concomitant statin, apharmaceutical composition comprising an anti-proprotein convertasesubtilisin/kexin type 9 (anti-PCSK9) antibody or antigen-binding proteinat a dose of about 150 mg every 4 weeks for at least 3 doses, therebytreating the hypercholesterolemia in the subject.

In some embodiments, the invention relates to a method for treating aform of hypercholesterolemia that is not Familial Hypercholesterolemiain a subject in need thereof comprising administering to the subject apharmaceutical composition comprising an anti-proprotein convertasesubtilisin/kexin type 9 (anti-PCSK9) antibody or antigen-binding proteinat a dose of about 150 mg every 4 weeks for at least 3 doses, therebytreating the form of hypercholesterolemia that is not FamilialHypercholesterolemia in the subject.

Thus, the methods of the present invention, according to certainembodiments, comprise administering a pharmaceutical compositioncomprising an anti-PCSK9 antibody to a subject, in the absence of astatin.

The methods of the present invention, according to certain embodiments,also comprise administering a pharmaceutical composition comprising ananti-CPSK9 inhibitor to a subject in combination with another non-statinlipid lowering agent.

Lipid lowering agents include, for example, agents which inhibitcholesterol uptake and or bile acid re-absorption (such as ezetimibe);agents which increase lipoprotein catabolism (such as niacin); and/oractivators of the LXR transcription factor that plays a role incholesterol elimination such as 22-hydroxycholesterol.

In some embodiments, the subject was previously on a therapeutic regimenfor the treatment of hypercholesterolemia prior to administration of thepharmaceutical composition of the invention. For example, a patient whohas previously been diagnosed with hypercholesterolemia may have beenprescribed and was taking a stable therapeutic regimen of another drugprior to administration of a pharmaceutical composition comprising ananti-PCSK9 antibody.

In some embodiments, the subject was previously treated with a statin orother lipid lowering agent prior to treatment with a PCSK9 inhibitordescribed herein. In other embodiments, the subject has not beenpreviously treated with a statin or other lipid lowering agent.

Exemplary Embodiments

In one aspect the present disclosure provides, a method for reducinglow-density lipoprotein cholesterol (LDL-C) in a subject in needthereof, the method comprising administering to the subject, who is nottaking a concomitant statin, a pharmaceutical composition comprising ananti-proprotein convertase subtilisin/kexin type 9 (anti-PCSK9) antibodyor antigen-binding protein at a dose of about 150 mg every 4 weeks forat least 3 doses, thereby reducing the LDL-C in the subject.

In certain embodiments, the antibody or antigen-binding fragment thereofcomprises the heavy and light chain complementarity determining regions(CDRs) of a heavy chain variable region/light chain variable region(HCVR/LCVR) amino acid sequence pair selected from the group consistingof SEQ ID NOs: 1/6 and 11/15. In certain embodiments, the antibody orantigen-binding fragment thereof comprises heavy and light chain CDRamino acid sequences having SEQ ID NOs:12, 13, 14, 16, 17, and 18. Incertain embodiments, the antibody or antigen-binding fragment thereofcomprises an HCVR having the amino acid sequence of SEQ ID NO:11 and anLCVR having the amino acid sequence of SEQ ID NO:15. In certainembodiments, the antibody or antigen-binding fragment thereof comprisesheavy and light chain CDR amino acid sequences having SEQ ID NOs:2, 3,4, 7, 8, and 10. In certain embodiments, the antibody or antigen-bindingfragment thereof comprises an HCVR having the amino acid sequence of SEQID NO:1 and an LCVR having the amino acid sequence of SEQ ID NO:6.

In certain embodiments, the antibody or antigen-binding fragment thereofbinds to the same epitope on PCSK9 as an antibody comprising heavy andlight chain CDR amino acid sequences having SEQ ID NOs:12, 13, 14, 16,17, and 18; or SEQ ID NOs: 2, 3, 4, 7, 8, and 10.

In certain embodiments, the antibody or antigen-binding fragment thereofcompetes for binding to PCSK9 with an antibody comprising heavy andlight chain CDR amino acid sequences having SEQ ID NOs:12, 13, 14, 16,17, and 18; or SEQ ID NOs: 2, 3, 4, 7, 8, and 10.

In certain embodiments, the subject has a form of hypercholesterolemiathat is not Familial Hypercholesterolemia (nonFH). In certainembodiments, the subject has heterozygous Familial Hypercholesterolemia(heFH). In certain embodiments, the diagnosis of heFH is made either bygenotyping or clinical criteria. In certain embodiments, the clinicalcriteria is either the Simon Broome Register Diagnostic Criteria forHeterozygous Familial Hypercholesterolemia, or the WHO/Dutch LipidNetwork criteria with a score >8.

In certain embodiments, the subject is on a non-statin lipid-loweringagent before and/or during administration of the antibody orantigen-binding protein. In certain embodiments, the non-statinlipid-lowering agent is selected from the group consisting of:ezetimibe, a fibrate, fenofibrate, niacin, an omega-3 fatty acid, and abile acid resin. In certain embodiments, the non-statin lipid-loweringagent is ezetimibe or fenofibrate.

In certain embodiments, the subject is not on a non-statinlipid-lowering agent before and/or during administration of the antibodyor antigen-binding protein.

In certain embodiments, the antibody or antigen binding protein isadministered subcutaneously.

In certain embodiments, the dose of about 150 mg every 4 weeks ismaintained if the subject's LDL-C measured after 4 or more does is <70mg/dL. In certain embodiments, the dose of about 150 mg every 4 weeks isdiscontinued if the subject's LDL-C measured after 4 or more dosesis >70 mg/dL, and the antibody or antigen binding protein issubsequently administered to the subject at dose of about 150 mg every 2weeks.

In another aspect the present disclosure provides, a method for treatinghypercholesterolemia in a subject in need thereof, the method comprisingadministering to the subject, who is not taking a concomitant statin, apharmaceutical composition comprising an anti-proprotein convertasesubtilisin/kexin type 9 (anti-PCSK9) antibody or antigen-binding proteinat a dose of about 150 mg every 4 weeks for at least 3 doses, therebytreating the hypercholesterolemia in the subject.

In certain embodiments, the antibody or antigen-binding fragment thereofcomprises the heavy and light chain complementarity determining regions(CDRs) of a heavy chain variable region/light chain variable region(HCVR/LCVR) amino acid sequence pair selected from the group consistingof SEQ ID NOs: 1/6 and 11/15. In certain embodiments, the antibody orantigen-binding fragment thereof comprises heavy and light chain CDRamino acid sequences having SEQ ID NOs:12, 13, 14, 16, 17, and 18. Incertain embodiments, the antibody or antigen-binding fragment thereofcomprises an HCVR having the amino acid sequence of SEQ ID NO:11 and anLCVR having the amino acid sequence of SEQ ID NO:15. In certainembodiments, the antibody or antigen-binding fragment thereof comprisesheavy and light chain CDR amino acid sequences having SEQ ID NOs:2, 3,4, 7, 8, and 10. In certain embodiments, the antibody or antigen-bindingfragment thereof comprises an HCVR having the amino acid sequence of SEQID NO:1 and an LCVR having the amino acid sequence of SEQ ID NO:6.

In certain embodiments, the antibody or antigen-binding fragment thereofbinds to the same epitope on PCSK9 as an antibody comprising heavy andlight chain CDR amino acid sequences having SEQ ID NOs:12, 13, 14, 16,17, and 18; or SEQ ID NOs: 2, 3, 4, 7, 8, and 10.

In certain embodiments, the antibody or antigen-binding fragment thereofcompetes for binding to PCSK9 with an antibody comprising heavy andlight chain CDR amino acid sequences having SEQ ID NOs:12, 13, 14, 16,17, and 18; or SEQ ID NOs: 2, 3, 4, 7, 8, and 10.

In certain embodiments, the subject has a form of hypercholesterolemiathat is not Familial Hypercholesterolemia (nonFH). In certainembodiments, the subject has heterozygous Familial Hypercholesterolemia(heFH). In certain embodiments, the diagnosis of heFH is made either bygenotyping or clinical criteria. In certain embodiments, the clinicalcriteria is either the Simon Broome Register Diagnostic Criteria forHeterozygous Familial Hypercholesterolemia, or the WHO/Dutch LipidNetwork criteria with a score >8.

In certain embodiments, the subject is on a non-statin lipid-loweringagent before and/or during administration of the antibody orantigen-binding protein. In certain embodiments, the non-statinlipid-lowering agent is selected from the group consisting of:ezetimibe, a fibrate, fenofibrate, niacin, an omega-3 fatty acid, and abile acid resin. In certain embodiments, the non-statin lipid-loweringagent is ezetimibe or fenofibrate.

In certain embodiments, the subject is not on a non-statinlipid-lowering agent before and/or during administration of the antibodyor antigen-binding protein.

In certain embodiments, the antibody or antigen binding protein isadministered subcutaneously.

In certain embodiments, the dose of about 150 mg every 4 weeks ismaintained if the subject's LDL-C measured after 4 or more does is <70mg/dL. In certain embodiments, the dose of about 150 mg every 4 weeks isdiscontinued if the subject's LDL-C measured after 4 or more dosesis >70 mg/dL, and the antibody or antigen binding protein issubsequently administered to the subject at dose of about 150 mg every 2weeks.

In another aspect the present disclosure provides, a method for treatinga form of hypercholesterolemia that is not Familial Hypercholesterolemiain a subject in need thereof, the method comprising administering to thesubject a pharmaceutical composition comprising an anti-proproteinconvertase subtilisin/kexin type 9 (anti-PCSK9) antibody orantigen-binding protein at a dose of about 150 mg every 4 weeks for atleast 3 doses, thereby treating the form of hypercholesterolemia that isnot Familial Hypercholesterolemia in the subject.

In certain embodiments, the antibody or antigen-binding fragment thereofcomprises the heavy and light chain complementarity determining regions(CDRs) of a heavy chain variable region/light chain variable region(HCVR/LCVR) amino acid sequence pair selected from the group consistingof SEQ ID NOs: 1/6 and 11/15. In certain embodiments, the antibody orantigen-binding fragment thereof comprises heavy and light chain CDRamino acid sequences having SEQ ID NOs:12, 13, 14, 16, 17, and 18. Incertain embodiments, the antibody or antigen-binding fragment thereofcomprises an HCVR having the amino acid sequence of SEQ ID NO:11 and anLCVR having the amino acid sequence of SEQ ID NO:15. In certainembodiments, the antibody or antigen-binding fragment thereof comprisesheavy and light chain CDR amino acid sequences having SEQ ID NOs:2, 3,4, 7, 8, and 10. In certain embodiments, the antibody or antigen-bindingfragment thereof comprises an HCVR having the amino acid sequence of SEQID NO:1 and an LCVR having the amino acid sequence of SEQ ID NO:6.

In certain embodiments, the antibody or antigen-binding fragment thereofbinds to the same epitope on PCSK9 as an antibody comprising heavy andlight chain CDR amino acid sequences having SEQ ID NOs:12, 13, 14, 16,17, and 18; or SEQ ID NOs: 2, 3, 4, 7, 8, and 10.

In certain embodiments, the antibody or antigen-binding fragment thereofcompetes for binding to PCSK9 with an antibody comprising heavy andlight chain CDR amino acid sequences having SEQ ID NOs:12, 13, 14, 16,17, and 18; or SEQ ID NOs: 2, 3, 4, 7, 8, and 10.

In certain embodiments, the subject has a form of hypercholesterolemiathat is not Familial Hypercholesterolemia (nonFH). In certainembodiments, the subject has heterozygous Familial Hypercholesterolemia(heFH). In certain embodiments, the diagnosis of heFH is made either bygenotyping or clinical criteria. In certain embodiments, the clinicalcriteria is either the Simon Broome Register Diagnostic Criteria forHeterozygous Familial Hypercholesterolemia, or the WHO/Dutch LipidNetwork criteria with a score >8.

In certain embodiments, the subject is on a non-statin lipid-loweringagent before and/or during administration of the antibody orantigen-binding protein. In certain embodiments, the non-statinlipid-lowering agent is selected from the group consisting of:ezetimibe, a fibrate, fenofibrate, niacin, an omega-3 fatty acid, and abile acid resin. In certain embodiments, the non-statin lipid-loweringagent is ezetimibe or fenofibrate.

In certain embodiments, the subject is not on a non-statinlipid-lowering agent before and/or during administration of the antibodyor antigen-binding protein.

In certain embodiments, the antibody or antigen binding protein isadministered subcutaneously.

In certain embodiments, the dose of about 150 mg every 4 weeks ismaintained if the subject's LDL-C measured after 4 or more does is <70mg/dL. In certain embodiments, the dose of about 150 mg every 4 weeks isdiscontinued if the subject's LDL-C measured after 4 or more dosesis >70 mg/dL, and the antibody or antigen binding protein issubsequently administered to the subject at dose of about 150 mg every 2weeks.

In another aspect the present disclosure provides, a dosing regimen ofan anti-proprotein convertase subtilisin/kexin type 9 (anti-PCSK9)antibody or antigen-binding protein that maintains a constantlow-density lipoprotein cholesterol (LDL-C) lowering throughout theinterdosing interval in a human subject which, following administrationof the anti-PCSK9 antibody or antigen-binding protein thereof at a doseof about 150 mg every 4 weeks for at least 3 doses, has one or more ofthe properties selected from the group consisting of: (a) an area underthe plasma concentration versus time curve calculated using thetrapezoidal method from time zero to real time (AUC_(last)) from about250 mg·day/L to about 650 mg·day/L; (b) a maximum plasma concentrationobserved (C_(max)) from about 15 mg/L to about 33 mg/L; (c) a first timeto reach a maximum plasma concentration (t_(max)) of about 7 days; and(d) a time to reach terminal half life (t_(1/2) ^(z)) from about 5.5days to about 12 days.

In another aspect the present disclosure provides, a dosing regimen ofan anti-proprotein convertase subtilisin/kexin type 9 (anti-PCSK9)antibody or antigen-binding protein that maintains a constantlow-density lipoprotein cholesterol (LDL-C) lowering throughout theinterdosing interval in a human subject which, following administrationof the anti-PCSK9 antibody or antigen-binding protein thereof at a doseof about 150 mg every 4 weeks for at least 3 doses, has one or more ofthe properties selected from the group consisting of: (a) an area underthe plasma concentration versus time curve calculated using thetrapezoidal method from time zero to real time (AUC_(last)) from about150 mg·day/L to about 450 mg·day/L; (b) a maximum plasma concentrationobserved (C_(max)) from about 10.5 mg/L to about 24 mg/L; (c) a firsttime to reach a maximum plasma concentration (t_(max)) of about 7 days;and (d) a time to reach terminal half life (t_(1/2) ^(z)) from about 5days to about 9 days.

In another aspect the present disclosure provides, a method formaintaining constant low-density lipoprotein cholesterol (LDL-C)lowering throughout an interdosing interval in a subject, the methodcomprising administering to the subject, who is not taking a concomitantstatin, a pharmaceutical composition comprising an anti-proproteinconvertase subtilisin/kexin type 9 (anti-PCSK9) antibody orantigen-binding protein at a dose of about 150 mg every 4 weeks for atleast 3 doses.

In certain embodiments, the antibody or antigen-binding fragment thereofcomprises the heavy and light chain complementarity determining regions(CDRs) of a heavy chain variable region/light chain variable region(HCVR/LCVR) amino acid sequence pair selected from the group consistingof SEQ ID NOs: 1/6 and 11/15. In certain embodiments, the antibody orantigen-binding fragment thereof comprises heavy and light chain CDRamino acid sequences having SEQ ID NOs:12, 13, 14, 16, 17, and 18. Incertain embodiments, the antibody or antigen-binding fragment thereofcomprises an HCVR having the amino acid sequence of SEQ ID NO:11 and anLCVR having the amino acid sequence of SEQ ID NO:15. In certainembodiments, the antibody or antigen-binding fragment thereof comprisesheavy and light chain CDR amino acid sequences having SEQ ID NOs:2, 3,4, 7, 8, and 10. In certain embodiments, the antibody or antigen-bindingfragment thereof comprises an HCVR having the amino acid sequence of SEQID NO:1 and an LCVR having the amino acid sequence of SEQ ID NO:6.

In certain embodiments, the antibody or antigen-binding fragment thereofbinds to the same epitope on PCSK9 as an antibody comprising heavy andlight chain CDR amino acid sequences having SEQ ID NOs:12, 13, 14, 16,17, and 18; or SEQ ID NOs: 2, 3, 4, 7, 8, and 10.

In certain embodiments, the antibody or antigen-binding fragment thereofcompetes for binding to PCSK9 with an antibody comprising heavy andlight chain CDR amino acid sequences having SEQ ID NOs:12, 13, 14, 16,17, and 18; or SEQ ID NOs: 2, 3, 4, 7, 8, and 10.

In certain embodiments, the subject has a form of hypercholesterolemiathat is not Familial Hypercholesterolemia (nonFH). In certainembodiments, the subject has heterozygous Familial Hypercholesterolemia(heFH). In certain embodiments, the diagnosis of heFH is made either bygenotyping or clinical criteria. In certain embodiments, the clinicalcriteria is either the Simon Broome Register Diagnostic Criteria forHeterozygous Familial Hypercholesterolemia, or the WHO/Dutch LipidNetwork criteria with a score >8.

In certain embodiments, the subject is on a non-statin lipid-loweringagent before and/or during administration of the antibody orantigen-binding protein. In certain embodiments, the non-statinlipid-lowering agent is selected from the group consisting of:ezetimibe, a fibrate, fenofibrate, niacin, an omega-3 fatty acid, and abile acid resin. In certain embodiments, the non-statin lipid-loweringagent is ezetimibe or fenofibrate.

In certain embodiments, the subject is not on a non-statinlipid-lowering agent before and/or during administration of the antibodyor antigen-binding protein.

In certain embodiments, the antibody or antigen binding protein isadministered subcutaneously.

In certain embodiments, the dose of about 150 mg every 4 weeks ismaintained if the subject's LDL-C measured after 4 or more does is <70mg/dL. In certain embodiments, the dose of about 150 mg every 4 weeks isdiscontinued if the subject's LDL-C measured after 4 or more dosesis >70 mg/dL, and the antibody or antigen binding protein issubsequently administered to the subject at dose of about 150 mg every 2weeks.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods and compositions of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention.

Example 1: Generation of Human Antibodies to Human PCSK9

Human anti-PCSK9 antibodies were generated as described in U.S. Pat. No.8,062,640. The exemplary PCSK9 inhibitor used in the following Examplesis the human anti-PCSK9 antibody designated “alirocumab”. Alirocumab hasthe following amino acid sequence characteristics: heavy chain variableregion (HCVR) comprising SEQ ID NO:90; light chain variable domain(LCVR) comprising SEQ ID NO:92; heavy chain complementarity determiningregion 1 (HCDR1) comprising SEQ ID NO:76; HCDR2 comprising SEQ ID NO:78;HCDR3 comprising SEQ ID NO:80; light chain complementarity determiningregion 1 (LCDR1) comprising SEQ ID NO:84; LCDR2 comprising SEQ ID NO:86;and LCDR3 comprising SEQ ID NO:88.

Alirocumab is a fully human monoclonal antibody that binds proproteinconvertase subtilisin kexin type 9 (PCSK9). Proprotein convertasesubtilisin kexin type 9 belongs to the subtilisin family of serineproteases and is highly expressed in the liver. PCSK9 is involved inregulating the levels of the low-density lipoprotein receptor (LDLR)protein. In animals and humans, alirocumab reduces LDL-C levels incirculation. Alirocumab may be an effective treatment to lower LDL-C andto reduce the risk for cardiovascular disease.

Example 2: A Randomized, Partial Blind, 3 Parallel Groups Study of thePharmacodynamic Profile of Alirocumab Administered as Multiple 150 mgSubcutaneous Doses, Either Alone or on Top of Ezetimibe or FenofibrateAdministered as Multiple Oral Doses in Healthy Subjects Introduction

A phase 1 clinical trial was conducted to evaluate the pharmacodynamicsand safety of an anti-PCSK9 antibody, alirocumab, in healthy subjects.The primary objective of the study was to assess the pharmacodynamicprofile of alirocumab administered either alone or on top of ezetimibeor fenofibrate, based on low density lipoprotein cholesterol (LDL-C).The secondary objectives of the study were: 1) to assess thepharmacodynamic profile of alirocumab administered either alone or ontop of ezetimibe or fenofibrate, based on other lipid parameters, 2) toassess the pharmacokinetic profile of alirocumab administered eitheralone or on top of ezetimibe or fenofibrate, 3) to document exposure toezetimibe and fenofibrate, 4) to assess the effect of either ezetimibeor fenofibrate on PCSK9 levels, and 5) to assess the safety ofconcomitant administration of alirocumab and either ezetimibe orfenofibrate.

This phase 1 study was a randomized, partial blind, controlled studyconducted in 3 parallel groups, with a 4-week run-in period with eitherezetimibe or fenofibrate or placebo ezetimibe, followed by a 17 weektreatment period (i.e.: group A: alirocumab+ezetimibe placebo, group B:alirocumab+ezetimibe, group C: alirocumab+fenofibrate). The studyinvestigated the impact of combining 150 mg Q4W with ezetimibe (EZE) andfenofibrate (FENO) on the LDL-C lowering effect and on circulatinglevels of free PCSK9 and total alirocumab.

Subjects

The study evaluated 72 healthy subjects. The criteria for inclusionwere: healthy male or female subjects, aged 18 to 65 years old, withserum LDL-C levels >130 mg/dL not receiving lipid lowering therapy atscreening (Day −29), and serum LDL-C levels 100 mg/dL at the baselinecontrol on Day −1 (before initiation of alirocumab). (Note: forpractical reasons, the blood sample for the measure of baseline LDL-Cwas taken on D-1, to verify that subject had still a LDL-C level 100mg/dL before initiating treatment with alirocumab).

Seventy-two subjects were randomized into three groups, with 24 subjectsper group. Baseline characteristics for the subjects are shown in Table1.

TABLE 1 Patient characteristics at baseline (Day −29) AlirocumabAlirocumab Alirocumab 150 mg 150 mg 150 mg Q4W + placebo Q4W + EZE Q4W +FENO Treatment group (N = 24) (N = 24) (N = 24) Age (years), mean (SD)48.5 (12.8) 49.5 (10.7) 54.6 (7.6) Male (%) 46 46 42 Body mass index(kg/m²), 23.9 (2.0) 25.5 (2.7) 24.7 (2.5) mean (SD) Calculated LDL-C(mg/dL), 183.3 (38.7) 181.7 (37.1) 180.6 (31.3) mean (SD) Totalcholesterol (mg/dL), 264.5 (43.7) 250.6 (40.6) 263.7 (40.6) mean (SD)Apolipoprotein B (g/L), 1.3 (0.21) 1.3 (0.22) 1.3 (0.17) mean (SD)Non-HDL-C (mg/dL), 198.4 (40.6) 200.3 (39.8) 199.5 (31.7) mean (SD)HDL-C (mg/dL), 65.7 (12.4) 60.3 (13.1) 64.2 (15.5) mean (SD)Triglycerides (mg/dL), 78.8 (44.3-177.1) 95.7 (35.4-168.3) 94.8(53.1-194.9) median (range) Lipoprotein (a) (g/L), 0.27 (0.0-1.6) 0.33(0.0-1.6) 0.17 (0.0-1.5) median (range) Free PCSK9 (ng/L), 146.5 (54.3)150.7 (48.5) 152.1 (54.1) mean (SD) HDL-C, high-density lipoproteincholesterol; SD, standard deviation

Study Treatments

Alirocumab was formulated as a 150 mg/ml solution for injection.Alirocumab was administered subcutaneously in the abdomen. Alirocumabwas administered at a dose of 150 mg, 1 injection every four weeks, fora total of three injections.

Ezetimibe was formulated as a 10 mg over-encasulated tablet (andmatching placebo ezetimibe capsule). Ezetimibe was administered orally.Ezetimibe was administered at a dose of 10 mg, once daily administrationfor a total duration of 21 weeks (4 weeks run-in followed by 17 weeksafter initial administration of alirocumab).

Fenofibrate was formulated at a 160 mg coated tablet. Fenofibrate wasadministered orally during a meal. Fenofibrate was administered at adose of 160 mg, once daily administration for a total duration of 21weeks (4 weeks of run-in followed by 17 weeks after initialadministration of alirocumab).

The duration of treatment was as follows. Subjects received repeateddoses (daily doses) of ezetimibe or fenofibrate or placebo ezetimibe forthe duration of the run-in period (Days −28 to −1), and throughout thetreatment phase (Days 1 to 120), and repeated doses of alirocumab (onDays 1, 29, and 57) on top of continuing ezetimibe or fenofibrate orplacebo ezetimibe.

The duration of observation was up to a maximum of 25 weeks per subject(from screening to end-of-study [EOS] visit) which included a screeningperiod of 3 weeks, a run-in period of 4 weeks, a treatment period of 17weeks, and an EOS visit (a minimum of 4 days after the last dose withezetimibe, placebo or fenofibrate).

Criteria for Evaluation

The pharmacodynamic criteria were as follows. The primary endpoint waspercent change in calculated serum LDL-C from baseline (D-29). Thesecondary endpoints were absolute change from baseline in calculatedLDL-C, total cholesterol (TC), high density lipoprotein cholesterol(HDL-C), non-HDL-C, triglycerides (TG), apolipoprotein B (ApoB),apolipoprotein A1 (ApoA1), and lipoprotein a (Lp[a]) analyzed in percentand absolute change from baseline.

The following pharmacokinetic (PK) parameters were calculated foralirocumab, using non-compartmental methods: maximum serum concentrationobserved (C_(max)), serum concentrations just before treatmentadministration during repeated dosing (C_(trough)), serum concentrationobserved at Day 29 (CD29), area under the serum concentration versustime curve calculated using the trapezoidal method from time zero to thereal time (AUC_(last)), area under the serum concentration versus timecurve extrapolated to infinity (AUC), time to reach C_(max) (t_(max)),area under the serum concentration versus time curve calculated usingthe trapezoidal method from time zero to Day 14 (AUC_(0-D14)), areaunder the serum concentration versus time curve calculated using thetrapezoidal method from time zero to study Day 29 (AUC_(0-D28)),terminal half-life associated with the terminal slope (t_(1/2z)), timecorresponding to the last concentration above the limit ofquantification (t_(last)) apparent total body clearance of a drug fromthe serum (CL/F), distribution volume at the steady-state (V_(ss/F)),mean time a molecule remains in the body (MRT), and distribution volumein the terminal phase (V_(z/F)). Total serum alirocumab concentrations,total and free proprotein convertase subtilisin kexin type 9 (PCSK9)concentrations, and anti-alirocumab antibodies were measured.

Subjects were monitored for safety via adverse events (AEs; includinglocal tolerability) spontaneously reported by the subjects or observedby the Investigator, clinical laboratory evaluations (biochemistry,hematology, coagulation, and urinalysis), vital sign assessments (heartrate, systolic blood pressure, and diastolic blood pressure), bodyweight, physical examination, 12-lead electrocardiogram (ECG) automaticon-site recordings, urine drug screen; alcohol breath or plasma test;β-human chorionic gonadotrophin levels in females; and immunogenicity(anti-alirocumab antibody titers) assessments.

Pharmacodynamic Sampling Times

The blood sampling for lipid parameters (ie, LDL-C, TC, HDL-C,non-HDL-C, TG, ApoB, ApoA1, Lp[a]) were performed in the morning, in thefasted condition (at least 10-hours fasted and refrained from smoking),before any drug intake.

Blood samples were collected at screening; Day −1 of the run-in phase;and Days 8, 15, 22, 29±1, 57±1, 64±1, 71±1, 78±1, 85±1, 99±2, and 120±3of the treatment phase.

Pharmacokinetic Sampling Times and Bioanalytical Methods

Pharmacokinetic blood samples for assay of alirocumab and total and freePCSK9 were collected at Day −28 and Day −15±2 of the run-in phase (assayof total and free PCSK9 only); at Days 1, 8, 15, 22, 29, 57, 64, 71, 78,85, 99, 120 of the treatment phase; and the EOS visit.

Pharmacokinetic blood samples for assay of total and unconjugatedezetimibe as well as fenofibric acid were collected at Day −28, Day −15,and Day −1 of the run-in phase; and Days 1, 29, 57, and 64 of thetreatment phase.

Samples for the determination of anti-drug antibody (ADA) levels inserum were collected at Day −28 of the run-in phase; Days 29, 57, 85,and 120 of the treatment phase; and the EOS visit.

Serum concentrations of alirocumab were determined using a validatedenzyme-linked immunosorbent assay (ELISA) method with a lower limit ofquantification (LLOQ) of 0.078 μg/mL.

Plasma concentrations of total and unconjugated ezetimibe, andfenofibric acid were determined using validated liquid chromatographywith tandem mass spectrometry (LC-MS/MS) methods with lower limits ofquantification of 0.2 ng/mL, 0.04 ng/mL, and 49.9 ng/mL, respectively.

Anti-alirocumab antibody samples were analyzed using a validatedelectrochemiluminescence assay for the determination of anti-alirocumabantibody titers in acid-treated human serum. Based on the minimumdilution of the samples, the minimum titer for any anti-alirocumabantibody positive sample was 30. In neat serum samples, the validatedlower limit of detection was approximately 1.5 ng/mL.

Summary Population Characterisitics

The study population included 72 subjects (24 in each treatment group);there were 32 male and 40 female subjects aged between 21 and 65 years.

Pharmacodynamic Results

LDL-C declined similarly in the ezetimibe and fenofibrate groups duringthe run-in period (Day −29 to Day −1) reaching −19.8 (2.1)% and −25.9(3.2)%, in the ezetimibe and fenofibrate groups respectively on Day −1and remained stable in the placebo group (+1.6 (3.0)% on Day −1) (FIG. 2). After the run-in period, treatment with alirocumab administered fromDay 1, produced a further decline, greater in the ezetimibe andfenofibrate groups. The relative change in LDL-C was parallel in the 3treatment groups until Day 15, then from Day 15 a modest reincrease wasstarting in the fenofibrate group while this was more stable until Day22 in the ezetimibe group from which a slight reincrease was also seen.This trend was not observed in the alirocumab alone group in which thedecline in LDL-C was sustained. The 3rd administration of alirocumabproduced a further decline in LDL-C, compared to the decline observedafter the 1st dose, and a maximum effect was achieved after the 3rdadministration of alirocumab on Day 71 (14 days after administration) inall treatment groups (p<0.0001), whatever the baseline considered (Day−29 or Day −1), and with a similar behavior to that seen 21 days afterthe 1st administration in the ezetimibe and fenofibrate groups (FIGS. 2& 3 ; Table 2). Maximum decreases were −47.59%, −65.34% and −66.75% inthe alirocumab alone, ezetimibe and fenofibrate groups, respectively(change from Day −29 baseline). Using the change from Day −1 baseline,maximum decreases were −47.39%, −56.56% and −54.34% in the alirocumabalone, ezetimibe and fenofibrate groups, respectively. On Day 28 afterthe 3^(rd) administration of alirocumab (ie: Day 85), decreases were−47.03%, −49.57% and −43.17% for alirocumab administered either alone,or with ezetimibe or with fenofibrate respectively (change from Day −1baseline). See FIGS. 2 and 3 .

Overall treatment effect was significant (p<0.0001). Pairwisecomparisons showed that the difference between alirocumab alone andalirocumab coadministered with ezetimibe remained significant across alltime points (FIG. 2 ). The difference between alirocumab alone andalirocumab coadministered with fenofibrate remained also significantacross all time points, except on Day 99 (CI: −13.8174 to 0.1219%).

As shown in FIG. 4 , analysis of the difference between mean estimatesof alirocumab coadministered with ezetimibe versus alirocumab alone andalirocumab coadministered with fenofibrate versus alirocumab alone donot show any difference over the time course of the study between thefenofibrate and the ezetimibe groups. Relative changes in TC wereparallel to the change in LDL-C. Treatment effect were significant forboth TG and HDL-C. Compared to alirocumab alone, fenofibrate produced anincrease in HDL-C and a decrease in TG, that were sustained andsignificant over most time points, whereas compared to alirocumab alonechanges in the ezetimibe group were generally not significant.

Effects on other lipid parameters are summarized in Table 2. Table 2shows that: 1) mean percentage (SEM) reductions from the main Day −29baseline to Day 71 were −48.2 (2.3)% with alirocumab alone, and −65.3(2.0)% and −66.8 (2.7)% with alirocumab+EZE and +FENO, respectively; and2) mean percentage (SEM) LDL-C reductions from the Day −1 baseline toDay 71 were −47.4 (3.2)% with alirocumab alone, and −56.6 (2.5)% and−54.3 (3.5)% with alirocumab+EZE and +FENO, respectively.

TABLE 2 Mean (SEM) percent change in other lipid parameters from the Day−29 baseline and Day −1 baseline to Day 71 (14 days after the 3^(rd)alirocumab dose) Main baseline (Day −29) Additional baseline (Day −1)(From start of placebo, EZE or (From start of alirocumab injection; FENOrun-in period) placebo/EZE/FENO treatment continued) AlirocumabAlirocumab Alirocumab Alirocumab Alirocumab Alirocumab 150 mg 150 mg 150mg 150 mg 150 mg 150 mg Q4W + placebo Q4W + EZE Q4W + FENO Q4W + placeboQ4W + EZE Q4W + FENO Treatment group (N = 24) (N = 24) (N = 24) (N = 24)(N = 24) (N = 24) LDL-C −48.2 (2.3) −65.3 (2.0) −66.8 (2.7) −47.4 (3.2)−56.6 (2.5) −54.3 (3.5) Total cholesterol −31.6 (1.4) −45.7 (1.5) −46.1(1.9) −31.5 (2.6) −36.5 (1.4) −32.4 (2.2) Non-HDL-C −43.0 (1.7) −60.6(1.9) −64.4 (2.5) −43.0 (2.7) −51.9 (2.1) −50.5 (3.2) Apolipoprotein B−39.1 (1.5) −53.5 (1.8) −58.3 (2.1) −38.4 (2.4) −44.9 (2.0) −44.6 (2.5)HDL-C 3.3 (3.4) 5.4 (3.2) 12.3 (3.1) 3.6 (2.9) 6.4 (3.1) 8.7 (3.0)Triglycerides* 5.7 −13.8 −36.0 −3.9 −16.5 −3.5 (−48.5 to 266.7) (−53.4to 53.5) (−57.9 to 11.3) (−41.3 to 77.6) (−37.2 to 24.2) (−58.0 to 74.1)Lipoprotein (a)* −20.3 −27.0 −19.9 −11.7 −9.2 −20.4 (−63.2 to 33.3)(−71.4 to 35.8) (−57.6 to 38.3) (−58.8 to 160.9) (−67.0 to 66.7) (−56.8to 17.7) *Median (range)

FIGS. 5 (A-D) are a group of four graphs that show the mean levels offree PCSK9, comparing the three treatment groups together, and comparedwith percent changes in LDL-C from the Day −29 baseline, per treatmentgroup.

FIGS. 6 (A-C) are a group of three graphs that show percent changes inLDL-C from the Day −29 baseline, levels of free PCSK9, and totalalirocumab from Day 57 (time of 3^(rd) alirocumab injection) to Day 85(28 days after 3rd alirocumab injection).

During the placebo run-in period (i.e., prior to alirocumab treatment),FENO resulted in increased free PCSK9 levels from 152 to 217 ng/mL.Corresponding changes in free PCSK9 were 147 to 119 ng/mL in the placebogroup and 151 to 142 ng/mL in the EZE group (FIG. 5A).

A complete suppression of free PCSK9 followed the 1st and 3rd alirocumabinjections (no measure was done after the 2nd injection) (FIG. 5A).

After 7 to 15 days post-alirocumab injection, levels of free PCSK9 hadincreased again in the FENO group, and, to a lesser extent, in the EZEgroup, compared with the alirocumab alone group (FIG. 5A). Theseincreases corresponded with the modest decrease in efficacy observed inthe EZE and FENO groups (FIG. 5B-D; and close-up in FIG. 6A+B).

Corresponding to the changes in free PCSK9, alirocumab exposures werereduced in the presence of FENO (geometric mean ratio [95% confidenceinterval] versus alirocumab alone: 0.64 [0.53 to 0.77]) as well as inthe presence of EZE (0.85 [0.70 to 1.03] versus alirocumab alone) (FIG.6C).

Safety Results

There was no death or SAE during the study. The incidence of TEAEs wassimilar across the 3 treatment groups (50.0%, 58.3% and 50.0% in thealirocumab alone, ezetimibe and fenofibrate groups respectively). Only 1subject had a TEAE of severe intensity, recorded in the fenofibrategroup (renal colic, lasting about 11 hours, occurring 64 days after thelast administration of alirocumab, and considered not related to thetreatment). The most frequent TEAEs (ie. recorded in >2 subjects in anytreatment group) were headache (3/24, 5/24, and 2/24 subjects in thealirocumab alone, ezetimibe and fenofibrate groups respectively),nasopharyngitis (3/24, 4/24, and 4/24 subjects in the alirocumab alone,ezetimibe and fenofibrate groups respectively), influenza (2/24, 0/24,and 1/24 subjects in the alirocumab alone, ezetimibe and fenofibrategroups respectively), gastroenteritis viral (0/24, 0/24, and 2/24subjects in the alirocumab alone, ezetimibe and fenofibrate groupsrespectively), influenza-like illness (1/24, 3/24, and 1/24 subjects inthe alirocumab alone, ezetimibe and fenofibrate groups respectively),and abdominal pain (2/24, 1/24, and 1/24 subjects in the alirocumabalone, ezetimibe and fenofibrate groups respectively). All other TEAEswere sporadic among the 3 groups.

There were few PCSAs in vital signs, with low incidence in the 3treatment groups. There were few PCSAs in ECG parameters, however noQTc>450 ms (male) or QTc>470 ms (female) was recorded and noprolongation of QTc (defined as increase from baseline >60 ms) wasdetected. None of these abnormalities in vital signs or ECG parameterswere considered clinically significant.

There were few PCSAs in hematology and biochemistry parameters with lowincidence in the 3 treatment groups. None of these abnormalities wereconsidered clinically significant, except for creatinine protein kinase(CPK) increase >10 upper limit of normal (ULN) in 1 subject of thealirocumab alone group. This CPK increase recorded at the EOS visit wasconsidered related to physical exercise, and declared as an AE. Therewere no PCSAs in liver function tests.

There were no serious adverse events (AEs) or discontinuations due toAEs. Treatment-emergent adverse events (TEAEs) are summarized in Table3.

One subject in the alirocumab+FENO group had a TEAE of severe intensity(renal colic), which was not considered by the investigator to berelated to the study treatment.

No clinically significant changes in vital signs, electrocardiogram,hematologic or biochemical parameters were observed in this study. Onesubject in the alirocumab+placebo group displayed an increase increatine phosphokinase >10 times the upper limit of normal, which wasconsidered by the Investigator to be related to physical exercise (Table3).

TABLE 3 Safety summary Alirocumab Alirocumab Alirocumab 150 mg 150 mg150 mg Q4W + placebo Q4W + EZE Q4W + FENO Treatment group (N = 24) (N =24) (N = 24) Patients with any TEAEs, n (%) 12 (50.0) 14 (58.3) 12(50.0) Most frequent TEAEs (recorded in ≥2 subjects in any group), n (%)Headache 3 (12.5) 5 (20.8) 2 (8.3) Nasopharyngitis 3 (12.5) 4 (16.7) 4(16.7) Influenza 2 (8.3) 0 (0) 1 (4.2) Gastroenteritis viral 0 (0) 0 (0)2 (8.3) Influenza-like illness 1 (4.2) 3 (12.5) 1 (4.2) Abdominal pain 2(8.3) 1 (4.2) 1 (4.2)

Immunogenicity Results

Four out of 24 subjects (16.7%) in the alirocumab alone group, 6/24subjects (25%) in the ezetimibe group, and 7/24 (29.2%) subjects in thefenofibrate group were tested ADA positive with titers ranging between30 (minimum detectable titer) and 240. A single titer of 240 wasdetected in a subject in the ezetimibe group on Day 29, declining to atiter of 30 on Day 85. On Day 120, no ADA's were detectable in thissubject. All other measured ADA titers were low and between 30 and 120for all other ADA positive subjects.

Over all groups, 4 subjects showed ADA positive titers in the predosesamples. This suggests that these subjects exhibited a pretreatmentimmunoreactivity in the assay, and not necessarily a treatment-emergentADA response to the administration of study drug. Only 1 pretreatmentpositive subject (fenofibrate group) had increased titers inpostbaseline period, with a maximum titer of 120 on Day 29 and Day 120.

Pharmacokinetic (PK) Results

Descriptive statistics of PK parameters of alirocumab following Q4Wrepeated SC doses of 150 mg alirocumab are provided in the tables below(Tables 4-6) and in FIG. 7 .

TABLE 4 Mean ± SD (Geometric Mean) [CV %] of alirocumab PK parameters inserum on Day 1 Serum Alirocumab Mean ± SD Alirocumab AlirocumabAlirocumab (Geometric Mean) Q4W 150 mg Q4W 150 mg Q4W 150 mg [CV %] SC +Placebo SC + Fenofibrate SC + Ezetimibe N 24    24    24    C_(max) 20.4± 13.5 14.6 + 4.06 18.2 ± 5.68 (mg/l) (18.3) [66.2] (14.1) [27.7] (17.3)[31.2] C_(D29)* 6.06 ± 2.91 3.40 ± 2.05  4.04 ± 2.25 (mg/l) (5.45)[48.0] (2.73) [60.4] (3.49) [55.6] t_(max) ^(a) 7.00 7.00 7.00 (day)(6.96-7.01) (6.97-7.01) (6.97-7.19) AUC_(last) 326 ± 125 233 ± 75.5  274± 87.4 (mg · day/l) (306) [38.4] (221) [32.3] (261) [31.8] AUC_(0-D28)**326 ± 125 233 ± 75.5  274 ± 87.4 (mg · day/l) (306) [38.4] (221) [32.3](261) [31.8] AUC 357 ± 210 241 ± 84.6  291 ± 92.9 (mg · day/l) (318)[58.9]^(b) (227) [35.0]^(c) (277) [31.9]^(c) AUC_(Ext)*** 25 ± 11 15 ±9  17 ± 11 (%) (22) [44.7] (12) [57.6] (15) [62.6] ^(a)Median(Min-Max)^(b)n = 8, ^(c)n = 18, *Concentration in serum on study day 29 (28 daysafter administration) **Partial AUC calculated between study days 1 and29 (PK time zero to Day 28) ***percentage of extrapolation of AUC

TABLE 5 Mean ± SD (Geometric Mean) [CV %] of alirocumab PK parameters inserum on Day 57, after the 3^(rd) alirocumab administration SerumAlirocumab Mean ± SD Alirocumab Alirocumab Alirocumab (Geometric Mean)Q4W 150 mg Q4W 150 mg Q4W 150 mg [CV %] SC + Placebo SC + FenofibrateSC + Ezetimibe N 24   24   24   C_(max) 24.3 ± 8.61 17.1 ± 6.66 21.9 ±8.91 (mg/l) (22.9) [35.5] (15.9) [38.9] (20.5) [40.6] C_(D29)* 7.07 ±4.66 4.08 ± 3.18 5.08 ± 3.26 (mg/l) (6.00) [66.0] (2.89) [77.9] (4.02)[64.3] t_(max) ^(a)  7.00  7.00  7.00 (day) (0.00-7.00) (6.97-7.99)(6.96-13.98) t_(last) ^(a) 69.00 63.00 64.99 (day) (42.00-77.05)(28.00-69.00) (41.95-70.07) t_(1/2z) 8.76 ± 3.12 7.07 ± 1.68 6.72 ± 1.56(day) (8.37) [35.7] (6.88) [23.8] (6.55) [23.3] AUC_(0-D28)** 445 ± 189292 ± 138 364 ± 143 (mg · day/l) (414) [42.3] (259) [47.3] (338) [39.4]CL/F 0.312 ± 0.124 0.595 ± 0.414 0.409 ± 0.176 (l/day) (0.285) [39.6](0.496) [69.6] (0.372) [43.0] Vss/F 5.46 ± 1.83 8.44 ± 4.18 6.27 ± 2.17(l) (5.19) [33.4] (7.62) [49.5] (5.91) [34.6] MRT 18.5 ± 3.84 15.6 ±2.78 16.1 ± 2.62 (day) (18.2) [20.7] (15.4) [17.8] (15.9) [16.3]^(a)Median (Min-Max) *Concentration in serum on study day 85 (28 daysafter the third administration) **Partial AUC calculated between studydays 57 and 85 (PK time zero to Day 28)

TABLE 6 Treatment period Parameter Comparison Estimate 90% CI Pointestimates of geometric mean ratio with 90% confidence interval fortreatment period [D1-D29] [D1-D29] C_(max) alirocumab + ezetimibe vs0.97 (0.82 to 1.14) alirocumab alone alirocumab + fenofibrate vs 0.78(0.66 to 0.92) alirocumab alone AUC alirocumab + ezetimibe vs 0.86 (0.67to 1.11) alirocumab alone alirocumab + fenofibrate vs 0.73 (0.57 to0.95) alirocumab alone AUC_(last) alirocumab + ezetimibe vs 0.88 (0.76to 1.03) alirocumab alone alirocumab + fenofibrate vs 0.74 (0.64 to0.86) alirocumab alone AUC_(0-D28) alirocumab + ezetimibe vs 0.88 (0.76to 1.03) alirocumab alone alirocumab + fenofibrate vs 0.74 (0.64 to0.86) alirocumab alone Point estimates of geometric mean ratio with 90%confidence interval for treatment period [D57-D126] C_(max) alirocumab +ezetimibe vs 0.92 (0.78 to 1.09) alirocumab alone alirocumab +fenofibrate vs 0.71 (0.60 to 0.84) alirocumab alone AUC_(0-D28)alirocumab + ezetimibe vs 0.85 (0.70 to 1.03) alirocumab alonealirocumab + fenofibrate vs 0.64 (0.53 to 0.77) alirocumab alonet_(1/2z) alirocumab + ezetimibe vs 0.80 (0.72 to 0.90) alirocumab alonealirocumab + fenofibrate vs 0.83 (0.74 to 0.93) alirocumab alone

After the 1^(st) injection, alirocumab C_(max) values were similar whencomparing alirocumab+ezetimibe versus alirocumab alone with a pointestimate of 0.97 (90% CI=0.82 to 1.14). Whereas a trend for reducedAUC_(0-D28) with a point estimate of 0.88 (90% CI=0.76 to 1.03) wasseen. Alirocumab serum exposure was reduced when comparingalirocumab+fenofibrate versus alirocumab alone with point estimates of0.78 (90% CI=0.66 to 0.92) and 0.74 (90% CI=0.64 to 0.86) for C_(max)and AUC_(0-D28), respectively.

After the 3^(rd) injection, alirocumab C_(max) values were similar whencomparing alirocumab+ezetimibe versus alirocumab alone with a pointestimate of 0.92 (90% CI=0.78 to 1.09). AUC_(0-D28) was seen to bereduced with a point estimate of 0.85 (90% CI=0.70 to 1.03). A trend fora shorter half-life (6.72±1.56 versus 8.76±3.12 days) was seen in thealirocumab+ezetimibe versus alirocumab alone group with a point estimateof 0.80 (90% CI=0.72 to 0.90).

Alirocumab serum exposure remained reduced when comparingalirocumab+fenofibrate versus alirocumab alone with point estimates of0.71 (90% CI=0.60 to 0.84) and 0.64 (90% CI=0.53 to 0.77) for C_(max)and AUC_(0-D28), respectively. Mean half-lives were reduced with a pointestimate of 0.83 (90% CI=0.74 to 0.93) when comparingalirocumab+fenofibrate versus alirocumab alone group. Half-lives of7.07±1.68 days and 8.76±3.12 days were calculated foralirocumab+fenofibrate and alirocumab alone groups, respectively.

Conclusions

LDL-C reductions with EZE and FENO therapy alone observed during therun-in period were as expected.

Alirocumab 150 mg Q4W monotherapy resulted in LDL-C reductions of ˜48%(regardless of baseline) which were sustained over the 28-day dosinginterval. Combination of alirocumab with EZE or FENO resulted in greaterLDL-C reductions than with alirocumab alone: 1) maximum reductions fromthe main baseline, which included the 28-day run-in with placebo, EZE orFENO, were ˜65% with EZE or FENO; 2) corresponding reductions using theadditional baseline (from first alirocumab injection) were ˜55% with EZEor FENO.

Treatment with FENO alone, and to a lesser extent EZE, resulted inmodest increases in free PCSK9 as compared with placebo.

The slight decrease in efficacy observed from 14 to 28 days after dosingwith alirocumab in the FENO combination group may be the result ofreduced alirocumab exposure due to increase in free PCSK9 levelsobserved with FENO administration. This also seemed to be the case, to alesser extent, with the EZE combination.

Previous studies suggested that concomitant statin therapy reduces theduration of efficacy of alirocumab via increased target mediatedclearance, requiring every 2 weeks dosing to overcome this. This studysuggests that other LLTs (EZE, FENO) may have less of an impact onalirocumab duration of efficacy, and so could be utilized withlower/less frequent doses of alirocumab.

Effects on other lipid parameters were as expected based on previousexperience and a similar incidence of TEAEs was reported in all groups.

Results of this study suggest that EZE and FENO therapy results inmodest increases in free PCSK9 levels that may explain slightly greaterreductions in LDL-C with alirocumab administration as well as a modestreduction in the duration of this maximal effect. However, these dataindicate that, unlike in combination with statins, alirocumab 150 mgcould be dosed Q4W in the setting of monotherapy and in combination withnon-statin LLTs.

Maximum decreases were −47.59%, −65.34%, and −66.75% in the alirocumabalone, ezetimibe, and fenofibrate groups, respectively (change from Day−29 baseline). Administration of alirocumab 150 mg Q4W either alone oron top of ezetimibe (10 mg/day) or fenofibrate (160 mg/day) in healthysubjects produced a decline in LDL-C reaching −47.39%, −56.56%, and−54.34% in the alirocumab alone, ezetimibe, and fenofibrate groupsrespectively 14 days after the 3^(rd) administration of alirocumab, incomparison to pre-alirocumab baseline value. A reduction of −47.03%,−49.57% and −43.17% in the alirocumab alone, ezetimibe, and fenofibrategroups respectively was still observed 28 days after this 3^(rd)administration of alirocumab. This suggests a maintenance of the effectwhen alirocumab was administered alone, whereas a small change from themaximum effect was seen between 2 and 4 weeks post dose in bothcombination arms. The coadministration of alirocumab with eitherezetimibe or fenofibrate produced a similar decline in LDL-C.

When comparing alirocumab+ezetimibe versus alirocumab alone Cmax valueswere similar, with a non-significant trend towards lower AUC_(0-D28) inthe alirocumab+ezetimibe treatment group and therefore a fasterelimination in this group.

Alirocumab PK parameters were significantly reduced by thecoadministration of fenofibrate. A daily dose of 160 mg fenofibratereduced the exposure of alirocumab in serum as described above. Ananalysis of t_(max) median difference in treatment showed no difference.

The incidences and titers of ADAs were similar for the 3 treatmentgroups. The serum concentrations of alirocumab in ADA positive andnegative subjects were comparable in the 3 treatment groups.

Continuous exposure of ezetimibe or fenofibrate in plasma was confirmedthroughout the study period.

Alirocumab administered either alone or on top of ezetimibe orfenofibrate at the dose of 150 mg Q4W for 3 administrations in healthysubjects was well tolerated.

Example 3: A Randomized, Double-Blind, Placebo-Controlled,Parallel-Group Study Evaluating the Efficacy and Safety of Alirocumab inPatients with Primary Hypercholesterolemia not Treated with a StatinSelection of Dose

Based on the results of studies carried out with statin as backgroundtherapy, the Q2W dosing regimen is appropriate to maintain constantLDL-C lowering throughout the interdosing interval in statin-treatedpatients, with the maximum efficacy at 12 weeks provided by the 150 mgQ2W dosing. However, for many patients, the magnitude of effect observedwith the 150 mg Q2W dose may not be needed to achieve the target LDL-Cgoal, and starting with a lesser dose may be undertaken.

The 150 mg Q4W dosing regimen for alirocumab that will be evaluated inthis study is based on the longer duration of action observed inpatients not receiving concomitant statin. A statin-stimulated increasedproduction of PCSK9 may affect the duration of action of alirocumab,because higher rates of PCSK9 production may result in greatertarget-mediated clearance of the antibody. Compared to statins,ezetimibe and fibrates appear to have little or no effect on PCSK9levels, and a Q4W dosing regimen is expected to maintain sufficientLDL-C lowering throughout the interdosing interval in patients notreceiving a statin but receiving these lipid-lowering therapies.

Rationale for Protocol Design

The objective of the present study is to assess the efficacy and safetyof alirocumab 150 mg Q4W as a potential starting dose in patients nottreated with a statin. The current study will provide information on theefficacy, safety and tolerability for an every 4 week dosing regimen,and in patients receiving a background therapy of ezetimibe orfenofibrate or diet alone.

None of the patients selected in this study will receive a statin. Acomponent of the population in the current study is statin intolerantpatients.

Overall, a target of ⅔ of patients should receive a background therapywith ezetimibe or fenofibrate.

In this study of patients maintaining their background therapy ofezetimibe or fenofibrate or diet alone, who do not/cannot receive astatin, the choice of placebo as control over the double-blind treatmentperiod appears appropriate for the objectives of this study, since itwill provide the most robust assessment of efficacy and safety of 150 mgQ4W. A calibrator arm of alirocumab 75 mg Q2W will provide a bench markfor the starting dose.

To help adjust the dosing regimen of alirocumab to patients' needs, inthe phase 3 program, alirocumab is initiated in most studies with a doseexpected to provide a 50% reduction in LDL-C at steady state (75 mg Q2Wprogram). In all the studies with 75 mg Q2W as a starting dose, the doseis increased to 150 mg Q2W at Week 12, based on LDL-C level achieved atWeek 8. In the current study assessing 150 mg Q4W as a potentialstarting dose in non-statin treated patients, the up-titration, ifneeded, will also be performed at week 12, as in all other studies ofthe program. In the whole program, the primary efficacy is assessedafter titration has taken place, as needed. In this study, the primaryefficacy parameter will be assessed at week 24. The 12-week efficacyassessment (i.e., before up-titration) will be important to consider,and will therefore be analyzed as a key secondary endpoint.

A 24 week duration for the double-blind period is considered adequate toprovide safety information on a dosing regimen including 150 mg Q4W as astarting dose, as exposure to alirocumab (C_(max) and AUC) will be inbetween that observed with 75 mg Q2W and 150 mg Q2W, for which a largedatabase will be available, over a longer duration. In thepost-titration period, patients not reaching LDL-C goal will receive 150mg Q2W, as in the rest of the program. To obtain additional safety datawith this Q4W dosing, patients may participate in an optional open-labeltreatment period from week 24 to June 2016.

This specific study is undertaken to demonstrate the safety and thereduction of low-density lipoprotein cholesterol (LDL-C) with a regimenincluding alirocumab 150 mg Q4W as a starting dose, as add-on tonon-statin lipid modifying background therapy (ezetimibe or fenofibrate)or with diet alone in comparison with placebo in patients with primaryhypercholesterolemia not treated with a statin. The statin intolerantpopulation that is not at LDL-C goal on optimized LMT (ezetimibe orfenofibrate) or on diet alone represents a group with an identifiedunmet medical need that can be addressed by adding alirocumab to theirLDL-C lowering therapies. At the end of the 24-week double-blindtreatment period, background therapy can be adjusted as needed inpatients opting to enter the optional open-label treatment period.

Study Objectives

The primary objective of the study is to demonstrate the reduction oflow-density lipoprotein cholesterol (LDL-C) by a regimen including analirocumab starting dose of 150 mg Q4W as add-on to non-statin lipidmodifying background therapy or as monotherapy in comparison withplacebo in patients with primary hypercholesterolemia not treated with astatin.

The secondary objectives are: to evaluate the effect of alirocumab, with150 mg Q4W as starting dose, in comparison with placebo on other lipidparameters (e.g., Apolipoprotein B (ApoB), non-high density lipoproteincholesterol (non-HDL-C), Total-Cholesterol (TC), Lipoprotein (a)(Lp[a]), high-density lipoprotein cholesterol (HDL-C), Triglycerides(TG) and Apolipoprotein A-1 (Apo A-1) levels; to evaluate the safety andtolerability of alirocumab 150 mg Q4W; to evaluate the development ofanti-alirocumab antibodies; and to evaluate the pharmacokinetics (PK) ofalirocumab 150 mg Q4W.

Other objectives are to evaluate efficacy and safety of a Q2W dosingregimen of mg alirocumab.

Study Design

This is a randomized, double-blind, placebo-controlled, parallel-group,multi-center phase 3 study. Randomization will be stratified accordingto the statin intolerant status and non-statin lipid modifyingbackground therapy. Only patients not receiving a statin will beincluded. Statin intolerant patients at moderate, high or very high CVrisk as defined below in the population section will represent a targetof approximately 50% of the study population. Statin intolerance isdefined for the trial as the inability to tolerate at least two statins:one statin at the lowest daily starting dose (defined as rosuvastatin 5mg, atorvastatin 10 mg, simvastatin 10 mg, lovastatin 20 mg, pravastatinmg, fluvastatin 40 mg, or pitavastatin 2 mg or as the lowest approveddaily dose by country specific labeling), and another stating at anydose, due to skeletal muscle-related symptoms, other than those due tostrain or trauma, such as pain, aches, weakness, or cramping, that beganor increased during statin therapy and stopped when statin therapy wasdiscontinued. Patients at moderate CV risk, not fulfilling the SIdefinition, will comprise the rest of the study population.

For the background therapy, a target of approximately ⅔ of patients willreceive a background therapy (fenofibrate or ezetimibe) and up to ⅓patients will be treated with diet alone.

The study comprises four periods:

-   -   1) A screening period of up to 3 weeks,    -   2) A double-blind, parallel-group treatment period of 24 weeks        over which patients will receive double-blind study treatment as        follows:    -   Alirocumab 150 mg subcutaneous every 4 weeks*    -   OR    -   Placebo for alirocumab subcutaneous every 2 weeks    -   OR    -   Alirocumab 75 mg subcutaneous every 2 weeks.        * the blind will be maintained in the alirocumab 150 mg every 4        weeks alternating with placebo SC Q4W.        At the week 12 visit, based on their LDL-C at week 8 and        baseline CV risk, patients will, in a blinded manner, either        continue receiving alirocumab 150 mg Q4W or 75 mg Q2W or will        have their dose up-titrated, as follows:        i) Patients with very high CV risk will, in a blinded manner,        either:    -   continue receiving alirocumab 150 mg Q4W or 75 mg Q2W from week        12 onwards until the last injection at week 22, if their week 8        LDL-C is <70 mg/dL (1.81 mmol/L) and they had at least a 30%        reduction of LDL-C from baseline at week 8; or    -   receive a dose that is up-titrated to alirocumab 150 mg Q2W from        Week 12 onwards until the last injection at week 22, if their        week 8 LDL-C is 70 mg/dL (1.81 mmol/L) or they do not have at        least 30% reduction of LDL-C from baseline at Week 8.        ii) Patients with high or moderate CV risk will, in a blinded        manner, either:    -   continue receiving alirocumab 150 mg Q4W or 75 mg Q2W from Week        12 onwards until the last injection at week 22, if their week 8        LDL-C is <100 mg/dL (2.59 mmol/L) and they had at least a 30%        reduction of LDL-C from baseline at week 8; or    -   receive a dose that is up-titrated to alirocumab 150 mg Q2W from        Week 12 onwards until the last injection at week 22, if their        week 8 LDL-C is 100 mg/dL (2.59 mmol/L) or they do not have at        least 30% reduction of LDL-C from baseline at Week 8.    -   3) A follow-up period of 8 weeks after the end of double-blind        treatment period.

Patients who are not eligible for the open-label treatment period willbe followed for a period of 8 weeks after the end of the double-blindtreatment period if they do not opt to or are not eligible toparticipate in the open-label treatment period. The 8-week follow-upperiod will not apply to patients who are eligible and choose to enrollin the open-label treatment period.

-   -   4) An optional open-label treatment period.

Patients who successfully complete the double-blind treatment periodwill be eligible (provided they have not experienced anytreatment-related AEs, or had significant protocol deviations) to enteran optional open-label treatment period.

Patients will receive alirocumab 150 mg Q4W at the start of theopen-label treatment period. The first injection during the open-labeltreatment period will be administered at the site at the week 24 visit(the first visit of the open-label treatment period).

From week 36 visit, based on LDL-C value at week 32, the Investigatorwill manage, based on his/her own judgment, adjustment of alirocumabdoses. At week 36, patients will either continue receiving alirocumab150 mg Q4W or will receive a dose that is up-titrated to alirocumab 150mg Q2W. Subsequent down titration to 150 mg Q4W will be allowed.

Although the background therapies should be maintained stable ifpossible, they might be adjusted based on Investigator judgment, inparticular in case of tolerability issue.

For adjustments based on LDL-C values, the Investigator can modifybackground therapy as needed. However, simultaneous adjustments inalirocumab dose and any LMT should be avoided.

Treatment for these patients will continue uninterrupted from the lastdose of study drug during the double-blind treatment period (week 22) toweek 24 (the first dose in the open-label treatment period) onward,until June 2016.

Duration of Study Participation

The study duration includes a screening period of up to 3-weeks, a24-week double-blind treatment period for efficacy and safetyassessment, and an 8-week post-treatment follow-up period for patientswho are not eligible for the open-label treatment period after the lastvisit of the DBTP. Thus the study duration per patient is about 35weeks+an optional open-label treatment period. The 8-week follow-upperiod will not apply to patients who are eligible and choose to enrollin the open-label treatment period. Patients who successfully completethe double-blind treatment period will be eligible (provided they havenot experienced any treatment-related AEs, or had significant protocoldeviations) to enter an optional open-label treatment period.

Selection of Patients

Patients meeting all of the following criteria will be considered forenrollment into the study. Patients with Primary hypercholesterolemia(heFH or non-FH) receiving fenofibrate or ezetimibe or diet alone. Onlypatients not receiving a statin will be included in the study, whichcorrespond to patients: who are intolerant to statins* as defined belowwith moderate, high, or very high CV risk; or who are not fulfilling theSI definition. Only patients at moderate CV risk will be included inthis stratum.

* Statin intolerance is defined as the inability to tolerate at least 2statins: 1 statin at the lowest daily starting dose (defined asrosuvastatin 5 mg, atorvastatin 10 mg, simvastatin 10 mg, lovastatin 20mg, pravastatin 40 mg, fluvastatin 40 mg or pitavastatin 2 mg or as thelowest approved daily dose by country specific labeling), AND anotherstatin at any dose, due to skeletal muscle-related symptoms, other thanthose due to strain or trauma, such as pain, aches, weakness, orcramping, that began or increased during statin therapy and stopped whenstatin therapy was discontinued.

Moderate CV risk is defined as a calculated 10-year fatal CVD risk SCORE1 and <5% (ESC/EAS 2012).

High CV risk is defined as a calculated 10-year fatal CVD risk SCORE 5%(ESC/EAS 2012), or moderate chronic kidney disease (CKD), or type 1 ortype 2 diabetes mellitus without target organ damage, or heFH (NCEP-ATPIII, ESC/EAS 2012).

Very high CV risk is defined as a history of documented CHD, ischemicstroke, peripheral arterial disease (PAD), transient ischemic attack(TIA), abdominal aortic aneurysm, carotid artery occlusion >50% withoutsymptoms, carotid endarterectomy or carotid artery stent procedure,renal artery stenosis, renal artery stent procedure, type 1 or type 2diabetes mellitus with target organ damage (NCEP-ATP III, ESC/EAS 2012).

A documented history of CHD (includes 1 or more of the following): acuteMI, silent MI, unstable angina, coronary revascularization procedure(e.g., percutaneous coronary intervention [PCI] or coronary arterybypass graft surgery [CABG]), and clinically significant CHD diagnosedby invasive or non-invasive testing (such as coronary angiography,stress test using treadmill, stress echocardiography or nuclearimaging).

Patients who have met all the above inclusion criteria will be screenedfor exclusion criteria. Exclusion criteria for the double-blind periodare: patients defined as statin intolerant and very high CV risk withLDL-C<70 mg/dL (1.81 mmol/L) at the screening visit (Week-3, V1);patients defined as statin intolerant and high or moderate CV risk withLDL-C<100 mg/dL (<2.59 mmol/L) at the screening visit (Week-3, V1);patients not fulfilling the statin intolerant definition and who are atmoderate CV risk with LDL-C<100 mg/dL (<2.59 mmol/L), at the screeningvisit (Week-3, V1); patients with LDL-C≤160 mg/dL (≥4.1 mmol/L) at thescreening visit (Week-3, V1) if receiving diet only, whatever the statinintolerance status or if non fulfilling statin intolerance definition atmoderate CV risk and treated with ezetimibe or fenofibrate; with a10-year fatal CVD risk SCORE<1% (ESC/EAS 2011) at the screening visit(Week-3, V1); newly diagnosed (within 3 months prior to randomizationvisit [Week 0]) or poorly controlled (HbA1c>9%) diabetes; with use ofstatin, red yeast rice products, niacin or bile acid sequestrant within4 weeks of the screening visit (Week-3) or between screening andrandomization visits; not on a stable dose of ezetimibe or fenofibratefor at least 4 weeks, prior to the screening visit (Week-3, V1) orbetween screening and randomization visits; with use of fibrates, otherthan fenofibrate, within 4 weeks of the screening visit (Week-3, V1) orbetween screening and randomization visits; with use of nutraceuticalsor over-the-counter therapies known to affect lipids, at a dose/amountthat has not been stable for at least 4 weeks, prior to the screeningvisit (Week-3, V1) or between screening and randomization visits;planned to undergo scheduled PCI, CABG, carotid or peripheralrevascularization during the study; systolic blood pressure (BP)>160mmHg or diastolic BP>100 mmHg at screening (Week-3, V1) and/orrandomization (Week 0) visits; history of New York Heart AssociationClass III or IV heart failure within the past 12 months; history of aMI, unstable angina leading to hospitalization, CABG, PCI, uncontrolledcardiac arrhythmia, carotid surgery or stenting, stroke, transientischemic attack, carotid revascularization, endovascular procedure orsurgical intervention for peripheral vascular disease within 3 monthsprior to the screening visit (Week-3, V1); known history of hemorrhagicstroke; age <18 years or legal age of majority at the screening visit(Week-3, V1) whichever is older; patients not previously instructed on acholesterol-lowering diet prior to the screening visit (Week-3, V1);presence of any clinically significant uncontrolled endocrine diseaseknown to influence serum lipids or lipoproteins; history of bariatricsurgery within 12 months prior to the screening visit (Week-3, V1);unstable weight defined by a variation >5 kg within 2 months prior tothe screening visit (Week-3, V1); known history of homozygous FH; knownhistory of loss of function of PCSK9 (i.e., genetic mutation or sequencevariation); use of systemic corticosteroids, unless used as replacementtherapy for pituitary/adrenal disease with a stable regimen for at least6 weeks prior to randomization; history of cancer within the past 5years, except for adequately treated basal cell skin cancer, squamouscell skin cancer, or in situ cervical cancer; known history of apositive HIV test; patient who has taken any active investigationaldrugs within 1 month or 5 half-lives, whichever is longer; patient whohas been previously treated with at least one dose of alirocumab or anyother anti-PCSK9 monoclonal antibody in other clinical trials; use ofcontinuous hormone replacement therapy unless the regimen has beenstable in the past 6 weeks prior to the Screening visit (Week-3) and noplans to change the regimen during the study; patient who withdrawsconsent during the screening period (patient who is not willing tocontinue or fails to return); conditions/situations or laboratoryfindings such as: any clinically significant abnormality identified atthe time of screening that in the judgment of the Investigator or anysub-Investigator would preclude safe completion of the study orconstrain endpoints assessment such as major systemic diseases, patientswith short life expectancy, patients considered by the Investigator orany sub-Investigator as inappropriate for this study for any reason,e.g.: those deemed unable to meet specific protocol requirements, suchas scheduled visits, those deemed unable to administer or toleratelong-term injections as per the patient or the investigator,investigator or any sub-Investigator, pharmacist, study coordinator,other study staff or relative thereof directly involved in the conductof the protocol, etc, presence of any other conditions (e.g.,geographic, social . . . ) actual or anticipated, that the Investigatorfeels would restrict or limit the patient's participation for theduration of the study; laboratory findings during the screening period(not including randomization labs): positive test for Hepatitis Bsurface antigen or Hepatitis C antibody, positive serum or urinepregnancy (including Week 0) test in women of childbearing potential,triglycerides >400 mg/dL (>4.52 mmol/L) (1 repeat lab is allowed),eGFR<30 mL/min/1.73 m2 according to 4-variable MDRD Study equation(Calculated by central lab), ALT or AST>3×ULN (1 repeat lab is allowed),CPK>3×ULN (1 repeat lab is allowed), TSH<LLN or >ULN (for patients onthyroid replacement therapy see earlier exclusion criterion; knownhypersensitivity to monoclonal antibody or any component of the drugproduct; and pregnant or breast-feeding women (women of childbearingpotential not protected by highly-effective method(s) of birth control(as defined in the informed consent form and/or in a local protocoladdendum) and/or who are unwilling or unable to be tested for pregnancy)

Exclusion criteria for the open-label period are: significant protocoldeviation in the double-blind period based on the Investigator judgment,such as non-compliance by the patient; any patient who experienced anadverse event leading to permanent discontinuation from the double-blindperiod; patients having any new condition or worsening of existingcondition which in the opinion of the Investigator would make thepatient unsuitable for enrollment, or could interfere with the patientparticipating in or completing the study; known hypersensitivity tomonoclonal antibody or any component of the drug product; positivepregnancy test at last visit of the double-blind period (W24, Visit 11);and women of childbearing potential not willing to continuehighly-effective method(s) of birth control (as defined in the informedconsent form and/or in a local protocol addendum) and/or who areunwilling or unable to be tested for pregnancy.

Study Treatments

For the double-blind treatment period, the study treatment is a singleSC injection of 1 mL for a 75 or 150 mg dose of alirocumab or placeboprovided in an auto-injector, administered in the abdomen, thigh, orouter area of the upper arm.

During the double-blind treatment period (Week 0 to 24), eligiblepatients will be randomized to receive: alirocumab 150 mg subcutaneousevery 4 weeks alternating with placebo of alirocumab subcutaneous every4 weeks, or alirocumab 75 mg subcutaneous every 2 weeks, or placebosubcutaneous every 2 weeks.

Study drug will be administered by SC injection Q2W, starting at week 0and continuing up to the last injection (week 22), 2 weeks before theend of the double-blind treatment period.

At the week 12 visit, based on their LDL-C at week 8 and baseline CVrisk, patients will either continue receiving alirocumab 150 mg Q4W or75 mg Q2W or will have their dose up-titrated, as follows:

-   -   1) Patients with very high CV risk will, in a blinded manner,        either: continue receiving alirocumab 150 mg Q4W or 75 mg Q2W        from week 12 onwards until the last injection at week 22, if        their week 8 LDL-C is <70 mg/dL (1.81 mmol/L) and they had at        least a 30% reduction of LDL-C from baseline at week 8, or        receive a dose that is up-titrated to alirocumab 150 mg Q2W from        week 12 onwards until the last injection at week 22, if their        week 8 LDL-C is 70 mg/dL (1.81 mmol/L) or they do not have at        least a 30% reduction of LDL-C from baseline at week 8.    -   2) Patients with high or moderate CV risk, will, in a blinded        manner, either: continue receiving alirocumab 150 mg Q4W or        75Q2W from week 12 onwards until the last injection at week 22,        if their week 8 LDL-C is <100 mg/dL (2.59 mmol/L) and they had        at least a 30% reduction of LDL-C from baseline at week 8, or        receive a dose that is up-titrated to alirocumab 150 mg Q2W from        week 12 onwards until the last injection at week 22, if their        week 8 LDL-C is 100 mg/dL (2.59 mmol/L) or they do not have at        least a 30% reduction of LDL-C from baseline at week 8.

During the open-label treatment period (from week 24 to June 2016),eligible patients will receive: alirocumab 150 mg Q4W up to week 36; andfrom week 36, according to LDL-C measurement at week 32 and the judgmentof the investigator, patients will either: continue receiving alirocumab150 mg Q4W, or receive a dose that is up-titrated to alirocumab 150 mgQ2W from week 36 onwards until June 2016. Subsequent down titration to150 mg Q4W will be allowed.

During the open-label treatment period, all patients will receivealirocumab 150 mg Q4W from week 24 to week 36. At the week 36 visit,based on their LDL-C at week 32, patients will either continue toreceive alirocumab 150 mg Q4W or will have their dose up-titrated toalirocumab 150 mg Q2W at the judgment of the investigator. Subsequentdown titration to 150 mg Q4W will be allowed.

Although the background therapies should be maintained stable ifpossible, they might be adjusted based on Investigator judgment, inparticular in case of tolerability issue. For adjustments based on LDL-Cvalues, the Investigator can modify background therapy as needed.However, simultaneous adjustments in alirocumab dose and any LMT shouldbe avoided.

Assessment of Alirocumab

The primary efficacy endpoint will be the percent change in calculatedLDL-C from baseline to Week 24, which is defined as: 100×(calculatedLDL-C value at Week 24-calculated LDL-C value at baseline)/calculatedLDL-C value at baseline.

The baseline calculated LDL-C value will be the last LDL-C levelobtained before the first double-blind injection IMP. For patientsrandomized and not treated, the baseline value is defined as the lastavailable value obtained up to randomization.

The calculated LDL-C at Week 24 will be the LDL-C level obtained withinthe Week 24 analysis window.

The main secondary efficacy endpoint(s) are: The percent change incalculated LDL-C from baseline to Week 12: similar definition and rulesas for primary efficacy endpoint, except that the calculated LDL-C atWeek 12 will be the LDL-C level obtained within the Week 12 analysiswindow; the percent change in Apo B from baseline to Week 24. Samedefinition and rules as for to the primary endpoint; the percent changein non-HDL-C from baseline to Week 24. Same definition and rules as forto the primary endpoint; the percent change in TC from baseline to Week24. Same definition and rules as for to the primary endpoint; thepercent change in Apo B from baseline to Week 12. Same definition andrules as for to the percent change in calculated LDL-C from baseline toWeek 12; the percent change in non-HDL-C from baseline to Week 12. Samedefinition and rules as for to the percent change in calculated LDL-Cfrom baseline to Week 12; the percent change in TC from baseline to Week12. Same definition and rules as for to the percent change in calculatedLDL-C from baseline to Week 12; the proportion of patients reachingcalculated LDL-C<70 mg/di (1.81 mmol/L) at Week 24 for very high CV riskpatients or <100 mg/di (2.59 mmol/L) for other patients using definitionand rules used for the primary endpoint; the percent change in Lp(a)from baseline to Week 24; the percent change in HDL-C from baseline toWeek 24; the percent change in HDL-C from baseline to Week 12; thepercent change in Lp(a) from baseline to Week 12; the percent change infasting TG from baseline to Week 24; the percent change in fasting TGfrom baseline to Week 12; the percent change in Apo A-1 from baseline toWeek 24; and the percent change in Apo A-1 from baseline to Week 12.

Other secondary efficacy endpoints are: the proportion of patientsreaching LDL-C<70 mg/dL (1.81 mmol/L) for very high CV risk patients or<100 mg/dL (2.59 mmol/L) for other patients at Week 12; the proportionof patients with LDL-C<100 mg/dL (2.59 mmol/L) at Weeks 12 and 24whatever the CV risk patients; the proportion of patients with LDL-C<70mg/dL (1.81 mmol/L) at Weeks 12 and 24 for very high CV risk patients;the absolute change in LDL-C (mg/dL and mmol/L) from baseline to Weeks12 and 24; the change in ratio Apo B/Apo A-1 from baseline to Weeks 12and 24; the proportion of patients with Apo B<80 mg/dL (0.8 g/L) atWeeks 12 and 24; the proportion of patients with non-HDL-C<100 mg/dL(2.59 mmol/L) at Weeks 12 and 24; the proportion of very high CV riskpatients with LDL-C<70 mg/dL (1.81 mmol/L) and/or 50% reduction in LDL-C(if LDL-C≥70 mg/dL) at Week 12 and 24; the proportion of patientsachieving at least 50% reduction in LDL-C at Weeks 12 and 24.

The lipid parameters will be assessed as follows. Total-C, HDL-C, TG,Apo B, Apo A-1, and Lp (a) will be directly measured. LDL-C will becalculated using the Friedewald formula at all visits (except Week-1 andFollow-Up visit). If TG values exceed 400 mg/dL (4.52 mmol/L) then thelab will reflexively measure (via the beta quantification method) theLDL-C rather than calculating it. LDL-C will also be measured (via thebeta quantification method) at Week 0 and Week 24 in all patients.Non-HDL-C will be calculated by subtracting HDL-C from the total-C.Ratio Apo B/Apo A-1 will be calculated.

The clinical laboratory data consist of urinalysis and blood analysis,hematology (RBC count, red blood cell distribution width (RDW),reticulocyte count, hemoglobin, hematocrit, platelets, WBC count withdifferential blood count), standard chemistry (glucose, sodium,potassium, chloride, bicarbonate, calcium, phosphorous, urea nitrogen,creatinine, uric acid, total protein, LDH, albumin, γ GlutamylTransferase [γGT]), Hepatitis C antibody, liver panel (ALT, AST, ALP,and total bilirubin), and CPK. Some additional safety laboratoryparameters may be reflexively measured, based on actual data.

The following vital signs will be measured: heart rate, systolic anddiastolic BP in sitting position.

The ECG data will be measured.

Anti-alirocumab antibodies will be assessed and include the antibodystatus (positive/negative) and antibody titers.

The percent change in hs-CRP will be assessed from baseline to Week 12and Week 24.

The absolute change in HbA1c (%) will be assessed from baseline to Week12 and Week 24.

EQ-5D is a standardized measure of health status developed by theEuroQol Group in order to provide a simple, generic measure of healthfor clinical and economic appraisal. The EQ-5D as a measure of healthrelated quality of life, defines health in terms of 5 dimensions:mobility, self-care, usual activities, pain/discomfort,anxiety/depression. Each dimension can take one of three responses (3ordinal levels of severity): ‘no problem’ (1) “some problems” (2)“severe problems” (3). Overall health state is defined as a 5-digitnumber. Health states defined by the 5-dimensional classification can beconverted into corresponding index scores that quantify health status,where 0 represents ‘death’ and 1 represents “perfect health”. EQ-5Dvariables include response of each EQ-5D items, index score and changeof index score from baseline.

Pharmacokinetic variables include total serum alirocumab concentration.Total and free PCSK9 concentrations will be measured from the same PKsample.

An optional pharmacogenomic sub-study will be conducted to identifygenetic associations with clinical or biomarker response to PCSK9inhibition, hyperlipidemia, or CVD. If needed, samples may also be usedto identify markers associated with toxicity. Analyses may includesequence determination or single nucleotide polymorphisms (SNP) fromcandidate genes. Candidate genes may include (but are not limited to)PCSK9, Apo B and LDL-R. Genome-wide studies, including (but not limitedto) SNP analyses and/or genomic sequencing may also be performed.

Study Procedures

For all visits after Day 1/Week 0 (randomization visit), a timeframe ofa certain number of days will be allowed. The window period for allvisits until Week 24 is ±3 days and for the follow-up period it is ±7days. During the open label period, the visit window is ±5 days forvisit Week 28, 32, 36 & ±7 days for the other visits.

The blood sampling for determination of lipid parameters (i.e., total-C,LDL-C, HDL-C, TG, non-HDL-C, Apo B, Apo A-1, ratio Apo B/Apo A-1, Lp[a]) should be performed in the morning, in fasting condition (i.e.overnight, at least 10 to 12 hours fast and refrain from smoking).

The following laboratory data are collected: Hematology; Chemistry;Lipid panel 1: TC, calculated LDL-C, HDL-C, TG, non-HDL-C; Lipid panel2: ApoB, ApoA-1, ratio ApoB/ApoA-1, and Lp(a); Liver panel: in case oftotal bilirubin values above the normal range, differentiation intoconjugated and non-conjugated bilirubin will occur automatically;Creatine Phosphokinase (CPK); Hepatitis B surface antigen; Hepatitis Cantibody: positive tests will be confirmed with reflexive testing; Serumpregnancy test.

Urinalysis—dipstick will be performed and will assess for pH, specificgravity, and for the presence of blood, protein, glucose, ketones,nitrates, leukocyte esterase, uro-bilinogen and bilirubin. If thedipstick is abnormal then standard microscopy will be conducted.

All other blood parameters will also be measured during the study.Glycemic parameters (HbA1c and serum glucose) will be measured. Theblood sampling for inflammatory parameter, hs-CRP will be collectedperiodically throughout the study.

Serum samples for assessment of alirocumab concentration will beobtained periodically throughout the study. Blood samples will becollected before IMP injection for visits 3 (week 0), 4 (week 4), 5(week 8), 6 (week 9), 7 (week 10), 8 (week 11), 9 (week 12), 10 (week16) and 11 (week 24). Blood samples should be collected before IMPinjection.

Library (plasma and serum) samples will be collected periodicallythroughout the study. The first scheduled sample at randomization visitwill be obtained before IMP injection (predose). Library samples mayinclude the study of PCSK9 levels, PCSK9 function, effect(s) of PCSK9inhibition with a monoclonal antibody, lipoprotein sub-fractionation,and mechanisms of hyperlipidemia and heart disease. If needed, samplesmay also be used to identify markers associated with toxicity. Thelibrary samples will never be used for genomic analysis.

A general physical examination should be performed at various pointsthroughout the study.

BP should be measured in sitting position under standardized conditions,approximately at the same time of the day, on the same arm, with thesame apparatus (after the patient has rested comfortably in sittingposition for at least 5 minutes). Heart rate will be measured at thetime of the measurement of BP.

The 12-lead ECGs should be performed after at least 10 minutes rest andin the supine position.

Body weight should be obtained with the patient wearing undergarments orvery light clothing and no shoes, and with an empty bladder. Heightshould also be obtained.

Visit Schedule

Only patients who meet the inclusion criteria should be screened. Thescreening period will take place up to 3 weeks or 21 days (and as shortas possible, upon receipt of laboratory eligibility criteria) prior torandomization/Day 1 visit. The Screening Visit (Visit1/Week-3/Day −21 upto −8) will include: Assess inclusion/exclusion criteria; Obtain patientdemography—age, gender, race, and ethnicity; Obtain medical history(including menopausal status), surgical history, alcohol habits, andsmoking habits; Obtain family medical history (including risk factorsrelating to premature CHD (before 55 years of age in a male, 65 years ina female first degree relative), allergy and Type 2 diabetes); Documentprior medication history within the previous 12 weeks, especially forlipid modifying therapy (including statin) and nutraceutical productsthat may affect lipids (e.g., omega-3 fatty acids, plant stanols such asfound in Benecol, flax seed oil, psyllium); Record concomitantmedication; Get body weight and height measurements. Take vital signsincluding HR and BP; Perform physical examination.

The Injection training visit at Screening (Visit 2/Week-1/Day −7±3) willinclude the following: Assess inclusion/exclusion criteria; Collect AEs;Record concomitant medication; Take vital signs including HR and BP.

The Randomization visit (Visit 3/Week 0/Day 1+3) will include thefollowing: Assess Inclusion/Exclusion Criteria; Collect AEs; Recordconcomitant medication; Review patient's diet. Patient should be on aNCEP-ATPIII TLC diet or equivalent; Perform physical examination; Getbody weight measurement; Take vital signs including HR and BP;Urinalysis (dipstick and if abnormal then microscopy); Urine pregnancytest (women of childbearing potential only); Obtain fasting blood samplefor: Lipids: measure and/or calculation of total-C, LDL-C (calculatedand measured LDL), HDL-C, TG, non-HDL-C, Apo B, Apo A-1, ratio Apo B/ApoA-1, and Lp (a); hs-CRP; Library samples; Hematology: red blood cellcount including hematocrit, hemoglobin, red blood cell distributionwidth (RDW), reticulocyte count, WBC count with differential count andplatelets; Chemistry: glucose, sodium, potassium, chloride, bicarbonate,calcium, phosphorous, urea nitrogen, creatinine, uric acid, LDH, totalprotein, albumin, and yGT; Liver panel (ALT, AST, ALP, and totalbilirubin); CPK; Anti-alirocumab antibodies; Serum alirocumabconcentration (PK); and Genomic specimen collection.

Visit 4/Week 4, (Day 29±3) will include the following: Collect AEs;Record concomitant medication; Take vital signs including HR and BP;Data collection on IMP administration and IMP compliance check by reviewof diary; Obtain fasting blood sample for: Lipids: measure orcalculation of total-C, LDL-C, HDL-C, TG, non-HDL-C; Liver panel (ALT,AST, ALP, and total bilirubin); Serum alirocumab concentration (PK); andAnti-alirocumab antibodies.

Visit 5/Week 8 (Day 57±3) will include the following: Collect AEs;Record concomitant medication; Take vital signs including HR and BP;Data collection on IMP administration and IMP compliance check by reviewof diary; Obtain fasting blood sample for: Liver panel (ALT, AST, ALP,and total bilirubin), Lipids: measure or calculation of total-C, LDL-C,HDL-C, TG, non-HDL-C, Apo B, Apo A-1, ratio Apo B/Apo A-1, and Lp (a),Serum alirocumab concentration (PK), and Anti-alirocumab antibodies.

Visits 6, 7, 8/Week 9, 10, 11 (Day 64, 71, 78±3) will include thefollowing: Blood samples should be collected before IMP injection; Serumalirocumab concentration (PK); Lipids: measure or calculation oftotal-C, LDL-C, HDL-C, TG, non-HDL-C; and Concomitant medications.

Visit 9/Week 12 (Day 85±3) will include the following: Collect AEs;Record concomitant medication; Get body weight measurement; Take vitalsigns including HR and BP; Review patient's diet. Patient should be on aNCEP-ATPIII TLC diet or equivalent; Perform 12-lead ECG; EQ-5D patientquestionnaire; Urinalysis (dipstick and if abnormal then microscopy);Urine pregnancy test (women of childbearing potential only); Obtainfasting blood sample for: Lipids: measure and/or calculation of total-C,LDL-C, HDL-C, TG, non-HDL-C, Apo B, Apo A-1, ratio Apo B/Apo A-1, and Lp(a); Library samples; Hematology: red blood cell count includinghematocrit, hemoglobin, red blood cell distribution width (RDW),reticulocyte count, WBC count with differential count and platelets;Chemistry: glucose, sodium, potassium, chloride, bicarbonate, calcium,phosphorous, urea nitrogen, creatinine, uric acid, LDH, total protein,albumin, and yGT. HbA1c and hs-CRP; Liver panel (ALT, AST, ALP, andtotal bilirubin); CPK; Anti-alirocumab antibodies; Serum alirocumabconcentration (PK).

Visit 10/Week 16 (Day 113±3) will include the following: Collect AEs;Record concomitant medication; Get body weight measurement; Take vitalsigns including HR and BP; Perform 12-lead ECG; Data collection on IMPadministration and IMP compliance check by review of diary; EQ-5Dpatient questionnaire; Urinalysis (dipstick and if abnormal thenmicroscopy); Urine pregnancy test (females of childbearing potentialonly); Obtain fasting blood sample for: Lipids: measure and/orcalculation of total-C, LDL-C, HDL-C, TG, non-HDL-C, Apo B, Apo A-1,ratio Apo B/Apo A-1, and Lp (a); Hematology: red blood cell countincluding hematocrit, hemoglobin, red blood cell distribution width(RDW), reticulocyte count, WBC count with differential count andplatelets; Chemistry: glucose, sodium, potassium, chloride, bicarbonate,calcium, phosphorous, urea nitrogen, creatinine, uric acid, LDH, totalprotein, albumin, and yGT. HbA1c and hs-CRP; Liver panel (ALT, AST, ALP,and total bilirubin); CPK; Anti-alirocumab antibodies; and Serumalirocumab concentration (PK).

Visit 11/Week 24/End of double-blind period (Day 169±3) will include thefollowing: Collect AEs; Record concomitant medication; Get body weightmeasurement; Take vital signs including HR and BP; Perform 12-lead ECG;Perform physical examination; EQ-5D patient questionnaire; Reviewpatient's diet. Patient should be on a NCEP-ATPIII TLC diet orequivalent; Urinalysis (dipstick and if abnormal then microscopy); Urinepregnancy test (women of childbearing potential only); Obtain fastingblood sample for: Lipids: measure and/or calculation of total-C, LDL-C(calculated and measured LDL), HDL-C, TG, non-HDL-C, Apo B, Apo A-1,ratio Apo B/Apo A-1, and Lp (a); hs-CRP; Library samples; Hematology:red blood cell count including hematocrit, hemoglobin, red blood celldistribution width (RDW), reticulocyte count, WBC count withdifferential count and platelets; Chemistry: glucose, sodium, potassium,chloride, bicarbonate, calcium, phosphorous, urea nitrogen, creatinine,uric acid, total protein, albumin, and γGT; Hepatitis B and C AntibodyTest (with automatic confirmatory testing if positive); HbA1c; Liverpanel (ALT, AST, ALP, and total bilirubin); CPK; Anti-alirocumabantibodies; and Serum concentration alirocumab (PK).

The Follow Up Visit (Visit 12/Week 32/Day 225±7) will include thefollowing: Collect AEs; Record concomitant medication; Take vital signsincluding HR and BP; Perform physical examination (only in case ofclinically relevant abnormality at the end of treatment visit);Urinalysis (only in case of clinically relevant abnormal value at theend of treatment visit); Urine pregnancy test (women of childbearingpotential only); Obtain fasting blood sample for: Anti-alirocumabantibodies, Only in case of clinically relevant abnormal values forthese parameters at the end of treatment visit will the following beobtained at this visit: Hematology: red blood cell count includinghematocrit, hemoglobin, red blood cell distribution width (RDW),reticulocyte count, WBC count with differential count and platelets;Chemistry: glucose, sodium, potassium, chloride, bicarbonate, calcium,phosphorous, urea nitrogen, creatinine, uric acid, total protein, LDH,albumin, and yGT; Liver panel (ALT, AST, ALP, and total bilirubin); andCPK.

Open Label Treatment Period (Optional)

Patients who successfully complete the double-blind treatment periodwill be eligible (provided they have not experienced anytreatment-limiting non-skeletal muscle-related AEs, or had significantprotocol deviations) to enter an optional open-label treatment period.Treatment for these patients will continue uninterrupted from the lastdose of study drug during the double-blind treatment period (week 22) toweek 24 (the first dose in the open-label treatment period) onward,until June 2016.

At Visit 11/Week 24, patients will undergo end of double-blind treatmentperiod assessments and baseline open-label treatment period assessments,concurrently. Study site personnel should review treatment requirementsof the open-label treatment period with patients and remind patientsthat dosing in the open-label treatment period begins at this visit. Thefollowing information will be collected: Assess Exclusion Criteria foropen-label treatment period; All evaluations performed for the end ofthe double-blind treatment period are the same for the first visit ofthe open-label treatment period; eview patient's diet. Patient should beon a NCEP-ATPIII TLC diet or equivalent. If the patient is confirmedeligible (and in fasting conditions), the Investigator will start thenext study procedures: The first open-label IMP injection will takeplace, but only after the collection of the fasting blood samples andafter the assessment of all evaluations planned at that visit.

Visit 12/Week 28 will include the following: Collect AEs; Recordconcomitant medication; Urine pregnancy test; Review patient's diet.Patient should be on a NCEP-ATPIII TLC diet or equivalent; Datacollection on IMP administration and IMP compliance check by review ofdiary; and Obtain fasting blood sample for: Liver panel (ALT, AST, ALP,and total bilirubin).

Visit 13/Week 32 will include the following: Collect AEs; Recordconcomitant medication; Urine pregnancy test; Review patient's diet.Patient should be on a NCEP-ATPIII TLC diet or equivalent; Datacollection on IMP administration and IMP compliance check by review ofdiary; Obtain fasting blood sample for: Liver panel (ALT, AST, ALP, andtotal bilirubin); Lipids: measure or calculation of total-C, LDL-C,HDL-C, TG, non-HDL-C; HbA1c; Hematology: red blood cell count includinghematocrit, hemoglobin, red blood cell distribution width (RDW),reticulocyte count, WBC count with differential count and platelets;Chemistry: glucose, sodium, potassium, chloride, bicarbonate, calcium,phosphorous, urea nitrogen, creatinine, uric acid, LDH, total protein,albumin, and yGT; and CPK.

Visit 14/Week 36 will include the following: Collect Aes; Recordconcomitant medication; Get body weight measurement; Take vital signsincluding HR and BP; Perform physical examination; Review patient'sdiet. Patient should be on a NCEP-ATPIII TLC diet or equivalent; EQ-5Dpatient questionnaire; Urine pregnancy test (women of childbearingpotential only); Obtain fasting blood sample for: Lipids: measure and/orcalculation of total-C, LDL-C, HDL-C, TG, non-HDL-C, Apo B, Apo A-1,ratio Apo B/Apo A-1, and Lp (a); Liver panel (ALT, AST, ALP, and totalbilirubin); Anti-alirocumab antibodies.

Visits 15, 17, 19/Week 48, 72, 96 will include the following: CollectAEs; Record concomitant medication; Get body weight measurement; Takevital signs including HR and BP; Review patient's diet. Patient shouldbe on a NCEP-ATPIII TLC diet or equivalent; Perform physicalexamination; Data collection on IMP administration and IMP compliancecheck by review of diary and treatment kit; EQ-5D patient questionnaire;Urinalysis (dipstick and if abnormal then microscopy); Urine pregnancytest (women of childbearing potential only); Obtain fasting blood samplefor: Lipids: measure and/or calculation of total-C, LDL-C, HDL-C, TG,non-HDL-C; Hematology: red blood cell count including hematocrit,hemoglobin, red blood cell distribution width (RDW), reticulocyte count,WBC count with differential count and platelets; Chemistry: glucose,sodium, potassium, chloride, bicarbonate, calcium, phosphorous, ureanitrogen, creatinine, uric acid, total protein, albumin, and yGT; HbA1c;Liver panel (ALT, AST, ALP, and total bilirubin); CPK; andAnti-alirocumab antibodies.

Visits 16, 18, 20/Week 60, 84, 108 will include the following: CollectAEs; Record concomitant medication; Review patient's diet. Patientshould be on a NCEP-ATPIII TLC diet or equivalent; and Data collectionon IMP administration and IMP compliance check by review of diary andtreatment kit.

Visit 21/Week 120 or June 2016 whichever comes first (end of OLTPtreatment) will include the following: Collect AEs; Record concomitantmedication; Get body weight measurement; Review patient's diet. Patientshould be on a NCEP-ATPIII TLC diet or equivalent; Take vital signsincluding HR and BP; Perform 12-lead ECG; Perform physical examination;Data collection on IMP administration and IMP compliance check by reviewof diary and treatment kit accountability; EQ-5D patient questionnaire;Urinalysis (dipstick and if abnormal then microscopy); Urine pregnancytest (women of childbearing potential only); Obtain fasting blood samplefor: Lipids: measure and/or calculation of total-C, LDL-C, HDL-C, TG,non-HDL-C, Apo B, Apo A-1, ratio Apo B/Apo A-1, and Lp (a); Hematology:red blood cell count including hematocrit, hemoglobin, red blood celldistribution width (RDW), reticulocyte count, WBC count withdifferential count and platelets; Chemistry: glucose, sodium, potassium,chloride, bicarbonate, calcium, phosphorous, urea nitrogen, creatinine,uric acid, total protein, albumin, and yGT. HbA1c; Liver panel (ALT,AST, ALP, and total bilirubin); CPK; and Anti-alirocumab antibodies.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figure. Such modifications are intended to fall within thescope of the appended claims.

1-78. (canceled)
 79. A method for reducing low-density lipoproteincholesterol (LDL-C) in a subject in need thereof, the method comprising:(a) selecting a subject with primary hypercholesterolemia who is nottaking a statin; and (b) administering to the subject a pharmaceuticalcomposition comprising an anti-proprotein convertase subtilisin/kexintype 9 (anti-PCSK9) antibody or antigen-binding fragment thereof at adose of about 300 mg every 4 weeks for at least 3 doses in the absenceof a concomitant statin therapy, wherein the antibody or antigen-bindingfragment thereof comprises the heavy and light chain complementaritydetermining regions (CDRs) of a heavy chain variable region/light chainvariable region (HCVR/LCVR) amino acid sequence pair of SEQ ID NOs: 1/6,thereby reducing the LDL-C in the subject.
 80. The method of claim 79,wherein the antibody or antigen-binding fragment thereof comprises heavyand light chain CDR amino acid sequences having SEQ ID NOs:2, 3, 4, 7,8, and
 10. 81. The method of claim 80, wherein the antibody orantigen-binding fragment thereof comprises an HCVR having the amino acidsequence of SEQ ID NO:1 and an LCVR having the amino acid sequence ofSEQ ID NO:6.
 82. The method of claim 79, wherein the subject is on anon-statin lipid-lowering agent before and/or during administration ofthe antibody or antigen-binding protein.
 83. The method of claim 82,wherein the non-statin lipid-lowering agent is selected from the groupconsisting of: ezetimibe, a fibrate, fenofibrate, niacin, an omega-3fatty acid, and a bile acid resin.
 84. The method of claim 83, whereinthe non-statin lipid-lowering agent is ezetimibe or fenofibrate.
 85. Themethod of claim 79, wherein the antibody or antigen binding protein isadministered subcutaneously.
 86. The method of claim 79, wherein thesubject is not on a non-statin lipid-lowering agent before and/or duringadministration of the antibody or antigen-binding fragment thereof. 87.The method of claim 79, wherein the subject has a form ofhypercholesterolemia that is not Familial Hypercholesterolemia (nonFH).88. The method of claim 79, wherein the subject has heterozygousFamilial Hypercholesterolemia (heFH).
 89. The method of claim 88,wherein the diagnosis of heFH is made either by genotyping or clinicalcriteria.
 90. The method of claim 89, wherein the clinical criteria iseither the Simon Broome Register Diagnostic Criteria for HeterozygousFamilial Hypercholesterolemia, or the WHO/Dutch Lipid Network criteriawith a score >8.
 91. The method of claim 79, further comprising the stepof (c) administering to the subject one or more following doses of 300mg of the antibody or antigen-binding fragment thereof about every fourweeks if the LDL-C level in the subject after step (b) is lower than 70mg/dL, or administering one or more following doses of 150 mg of theantibody or antigen-binding fragment thereof about every two weeks ifthe LDL-C level in the subject after step (b) is greater than or equalto 70 mg/dL.
 92. The method of claim 79, further comprising the step ofmeasuring the LDL-C of the subject after step (a) and before step (b).93. The method of claim 92, wherein the LDL-C of the subject is measuredjust prior to the next scheduled dose of the antibody or antigen-bindingfragment thereof.
 94. The method of claim 91, further comprising thestep of measuring the LDL-C of the subject after step (b) and beforestep (c).
 95. The method of claim 94, wherein the LDL-C of the subjectis measured just prior to the next scheduled dose of the antibody orantigen-binding fragment thereof.